Near-infrared light-triggered NO release for spinal cord injury repair

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Science Advances  25 Sep 2020:
Vol. 6, no. 39, eabc3513
DOI: 10.1126/sciadv.abc3513
  • Fig. 1 Schematic diagram of NIR-controlled NO release for SCI repair.

    The NO delivery nanosystem was constructed as an UCNP core coated by zeolitic imidazolate framework material (ZIF-8) shell with NO photochemical donor (CysNO). UCNPs convert NIR light to blue-violet light, which can cleave the S─NO bond in CysNO for NO release. The pleiotropic effects of NO, including the suppression of gliosis and inflammation, the promotion of neuronal regeneration, and the protection of neurons from apoptosis, facilitate the growth of injured motor neuron axons in zebrafish, as well as the recovery of the motor functions in traumatic SCI rats.

  • Fig. 2 Synthesis and characterization of UCZN.

    (A) Preparation of UCZN. (B) TEM images of UCNPs, UCNPs@PVP, and UCNPs@PVP@ZIF-8@CysNO (UCZN). (C) XRD patterns of UCNPs (a) and UCNPs@PVP@ZIF-8 (UCZs) (b). (D) FTIR spectra of UCNPs@PVP, UCZs, and UCZN, with the characteristic peaks of RSNOs marked by the green circles. (E) Thermogravimetric curves of the product in each step, with the calculated difference values between them. (F) Fluorescence emission spectra of UCNPs and UV-Vis absorption of UCZN (wavelength from 300 to 440 nm). (G) Released amount of NO from UCZN [160 parts per million (ppm)] with or without NIR light activation. The amount of NO was calculated according to the standard curve (top left corner). NIR light (980 nm, 1.5 W/cm2) was switched on/off every minute. (H) NO release curves of UCZN at different concentrations upon NIR light stimulation (980 nm, 1.5 W/cm2) at room temperature during 1 week. The solid lines depict the rapid release of a large amount of NO within 1 day after activation by NIR light. The dotted lines indicate the continuous slow release of NO over the remaining 6 days.

  • Fig. 3 Spatiotemporally controlled release of NO and growth promotion in PC12 cells.

    (A) Confocal microscopy images of PC12 cells precultured with UCZN before and after NIR stimulation. PC12 cells were stained with NO green fluorescence probe (DAF-FM DA). UCZN converted the 980-nm light to blue-violet light. The orange square is the stimulated ROI by NIR light (980 nm, 1.5 W/cm2), and the blue square is the control ROI without 980-nm laser stimulation. The real-time curves of signal intensity in different ROIs were shown on the right (scale bar, 50 μm). (B) Confocal microscopy images of calcein-labeled PC12 cells after different treatments (varied concentrations of UCZN with or without NIR light) for 6 days (scale bar, 50 μm). (C and D) Trends of PC12 cell differentiation (n = 20, groups = 5, mean ± SD) and the neurite number (n = 5, groups = 6, mean ± SD), neurite length (n = 6, group = 1, mean ± SD), and growth ratio (n = 100, groups = 3, mean ± SD) of PC12 cells over 6 days. The level of growth was quantitatively divided into L0 to L6 according to the length of the neurites, and details are in Materials and Methods.

  • Fig. 4 Mechanism of neuronal modulation via UCZN.

    (A) DRG neurons precultured with UCZN (blue-violet) were stained with [Ca2+]in green fluorescence probe (Fluo-4 AM). The red square was set as the UCZN-activated ROI with 980-nm laser. Other ROIs (yellow, orange, and blue squares) were selected from DRG neurons, and real-time curves of signal intensity were shown on the right (scale bar, 50 μm). (B) [Ca2+]in imaging of DRG neurons transfected with GCaMP-X and cocultured with UCZN before and after NIR stimulation. The red square is the stimulated ROI with 980-nm laser irradiation (1.5 W/cm2). The real-time change of fluorescence intensity in the selected ROI is shown on the right (scale bar, 200 μm). (C) Differentiation and neurite growth status of PC12 cells in the presence of nonspecific calcium ion channel blocker LaCl3 under different treatments (scale bar, 50 μm). (D) Relevant statistics of cell differentiation (n = 20, groups = 5, mean ± SD), neurite number (n = 5, groups = 6, mean ± SD), and neurite length (n = 6, group = 1, mean ± SD) in comparison with the cases without LaCl3. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 5 UCZN repaired the injured spinal cord in zebrafish and Sprague-Dawley rats.

    (A) Fluorescent images of the spinal cord of zebrafish under different treatments (scale bar, 100 μm). The motor neuron axons are marked by red lines. (B) Average axon length. (C and D) Movement velocity and time of Sprague-Dawley rats in different groups (n = 3, mean ± SD). (E) H&E-stained sections of injured spinal cords without (a) or with (b) the UCNP and 980-nm treatment (scale bar, 2500 μm). Representative immunofluorescence images of GFAP, IBA1, GAP-43, and caspase-3 at both the lesion and the well region: without (c) or with (d) UCNPs +980 treatment (scale bar, 200 μm). Cell nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) in blue. The lesion and the well region were labeled in the H&E-stained sections with red and blue rectangles, respectively. (F) The lesion areas were analyzed based on H&E-stained sections (n = 3, mean ± SD). (G) Relative fluorescent areas of GFAP, IBA1, GAP-43, and caspase-3 were analyzed based on corresponding immunofluorescence images (n = 3, mean ± SD). *P < 0.05, **P < 0.01, and ***P < 0.001.

Supplementary Materials

  • Supplementary Materials

    Near-infrared light-triggered NO release for spinal cord injury repair

    Yaqin Jiang, Pengfei Fu, Yanyan Liu, Chaochao Wang, Peiran Zhao, Xu Chu, Xingwu Jiang, Wei Yang, Yelin Wu, Ya Wang, Guohua Xu, Jin Hu, Wenbo Bu

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