Research ArticleHEALTH AND MEDICINE

Highly bioactive zeolitic imidazolate framework-8–capped nanotherapeutics for efficient reversal of reperfusion-induced injury in ischemic stroke

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Science Advances  18 Mar 2020:
Vol. 6, no. 12, eaay9751
DOI: 10.1126/sciadv.aay9751
  • Fig. 1 Structural characterization of CeO2@ZIF-8 nanomaterials.

    (A) Schematic illustration for in situ synthetic approach of CeO2@ZIF-8 nanotherapeutics and its neuroprotective application mechanisms against reperfusion-induced injury in ischemic stroke. (B and C) SEM and TEM images of ZIF-8 (B) and CeO2@ZIF-8 nanomaterials (C). (D) HAADF-STEM (high-angle annular dark-field imaging–scanning TEM) image and linear TEM-EDS (energy-dispersive x-ray spectroscopy) image of CeO2@ZIF-8. (E) Elemental mapping of CeO2@ZIF-8. XRD pattern (F), UV-vis spectra (G), Raman spectra (H), and XPS spectra (I) of CeO2, ZIF-8, and CeO2@ZIF-8 nanomaterials. (J and K) XPS analysis of Zn 2p (J) and Ce 3d (K) spectra of ZIF-8, CeO2, and CeO2@ZIF-8 nanomaterials. a.u., arbitrary units.

  • Fig. 2 ROS scavenging by CeO2@ZIF-8 nanomaterials in vitro.

    (A) Schematic demonstration of ROS scavenging by CeO2@ZIF-8 nanomaterials in vitro. (B) In vitro antioxidant activity of CeO2, ZIF-8, and CeO2@ZIF-8 nanomaterials as determined by ABTS free radical scavenging assays. *P < 0.05, **P < 0.01, and ***P < 0.001. (C) Raman spectra of CeO2@ZIF-8 after reaction with H2O2 at various time points. (D) TEM image of CeO2@ZIF-8 and ZIF-8 after incubations in aqueous solution with H2O2 (5%) for 3 hours. (E) EPR spectra analysis of the OH scavenging by CeO2 (15 μg/ml), ZIF-8, and CeO2@ZIF-8 nanomaterials. OH was generated by the Fenton reaction with Fe2+/H2O2 system and detected by DMPO. (F) UV-vis spectra of salicylic acid (SA) after reaction with OH generated by the Fenton reaction with Fe2+/H2O2 system for 10 min. I: SA; II: SA + H2O2; III: SA + Fe2+; IV: SA + Fe2+/H2O2; V: SA + Fe2+/H2O2 + CeO2@ZIF-8 (10 μg/ml); VI: SA + Fe2+/H2O2 + CeO2@ZIF-8 (20 μg/ml). (G) EPR spectra analysis of O2 scavenging with CeO2, ZIF-8, and CeO2@ZIF-8 nanomaterials (15 μg/ml). O2 was generated by the reaction of xanthine and xanthine oxidase for 30 min and detected by the DHE probe. (H) Fluorescence spectra analysis of O2 scavenging with different concentrations of CeO2@ZIF-8.

  • Fig. 3 Protection of CeO2@ZIF-8 to PC12 cells against t-BOOH–induced oxidative damage.

    (A) The cell viability of PC12 cells cotreated with 20 μM t-BOOH and CeO2, ZIF-8, or CeO2@ZIF-8 nanomaterials for 48 hours. *P < 0.05 and **P < 0.01. (B) Annexin V–fluorescein isothiocyanate (FITC)/PI double staining to evaluate the reversal of t-BOOH–induced apoptosis by CeO2@ZIF-8. PC12 cells were treated with t-BOOH and various concentrations of CeO2@ZIF-8 for 48 hours. (C) CeO2@ZIF-8 reduces t-BOOH–induced PC12 cell apoptosis. PC12 cells were treated with t-BOOH and various concentrations of CeO2@ZIF-8 for 48 hours. (D) CeO2@ZIF-8 blocks t-BOOH (15 μM)–induced ROS generation in PC12 cells. (E) CeO2@ZIF-8 inhibits t-BOOH–induced mitochondrial fragmentation in PC12 cells. (F) Schematic demonstration of the transwell assay for the coculture of BBB model. (G) Penetrative capacity and ratio of CeO2@ZIF-8 across BBB. (H) Internalization of the penetrative CeO2@ZIF-8 in the lower chamber of PC12 cells after penetrating BBB for 24 hours.

  • Fig. 4 Endocytosis of CeO2@ZIF-8 in PC12 cells.

    (A) Schematic demonstration of endocytosis of CeO2@ZIF-8 in PC12 cells. (B) TEM image of CeO2@ZIF-8 internalized in PC12 cells. PC12 cells were incubated with CeO2@ZIF-8 (16 μg/ml) for 6 hours. (C) TEM images of CeO2@ZIF-8 in aqueous (I), PBS (pH 7.4; II), and PBS (pH 5.3 with lysozyme; III) after shaking for 12 hours. (D) Intracellular trafficking of C6-labeled CeO2@ZIF-8 (20 μg/ml; green fluorescence) in PC12 cells. The cells were stained with special fluorescent tracers: LysoTracker (red) and Hoechst 33342 (blue). (E) Quantitative analysis of cellular uptake of CeO2@ZIF-8 in PC12 cells by determination of fluorescence intensity. (F and G) Intracellular uptake inhibition by different endocytosis inhibitors. The cells were pretreated with different endocytosis inhibitors for 1 hour and then incubated with CeO2@ZIF-8 (10 μg/ml) for 2 hours at 37° or 4°C. The control group was incubated with CeO2@ZIF-8 at 37°C only. **P < 0.01 and ***P < 0.001.

  • Fig. 5 CeO2@ZIF-8 reduces infarct volume by reducing ROS-induced oxidative damage.

    (A) Representative images of TTC-stained brain slices after treatment with CeO2@ZIF-8 for 3 days in MCAO mice model (n = 4). (B) Corresponding infarct areas of different groups analyzed by ImageJ (n = 4). (C) Neurological scores of MCAO mice after treatment with CeO2@ZIF-8 for 3 days (n = 10). (D) Assessment of CeO2@ZIF-8 (0.4 mg/kg) internalization in the brain tissue of MCAO mice for 3 days using TEM imaging (n = 3). (E) Biodistribution of Ce in the mice main organs after intravenous injection with CeO2 and CeO2@ZIF-8 (n = 5) for 3 days. (F) Ce content in plasma versus time after intravenous injection with CeO2 and CeO2@ZIF-8 (n = 5). (G to J) Expression levels of superoxide anion (G), MDA (H), SOD (I), and GSH-Px (J) in the brain tissue of different treatment groups (n = 3). Significant difference between treatment and control groups is indicated at *P < 0.05, **P < 0.01, and ***P < 0.001 levels.

  • Fig. 6 Therapeutic effects of CeO2@ZIF-8 and its suppression on inflammation and immune response induced by reperfusion in ischemic stroke.

    Representative photomicrographs of H&E staining (A), Nissl staining (B), and TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling)–Hoechst costaining (C) of brain tissues from different treatment groups. (D) GFAP and Iba-1 expression in brain sections of MCAO mice from different treatment groups examined by immunohistochemical staining (n = 3). (E and F) The number of GFAP+ cells (E) and Iba-1+ cells (F) in brain sections of MCAO mice from different treatment groups. (G to I) Inflammatory factors of TNF-α (G), IL-1β (H), and IL-6 (I) in different treatment groups (n = 3) in the infarct part of the brain tissue. **P < 0.01 and ***P < 0.001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/12/eaay9751/DC1

    Fig. S1. Characterization of the synthetic nanomaterials.

    Fig. S2. Examination of free radical scavenging activity of the nanotherapeutics.

    Fig. S3. N2 adsorption-desorption isotherm and pore property analysis.

    Fig. S4. CeO2@ZIF-8 inhibits cell apoptosis and ROS overproduction induced by t-BOOH.

    Fig. S5. Change in cell microstructure and mice body weight.

    Fig. S6. Brain protection effects of CeO2@ZIF-8 in C57 MCAO mice.

    Fig. S7. Fluorescence imaging and biodistribution analysis of CeO2@ZIF-8 in vivo.

    Fig. S8. H&E staining of the heart, liver, spleen, lung, and kidney after treatment with CeO2@ZIF-8 for 3 days in MCAO mice model.

    Fig. S9. Toxicity evaluation of CeO2@ZIF-8 in vivo.

    Table S1. Pharmacokinetic parameters of CeO2 nanopolyhedra and CeO2@ZIF-8 composite nanomaterials.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Characterization of the synthetic nanomaterials.
    • Fig. S2. Examination of free radical scavenging activity of the nanotherapeutics.
    • Fig. S3. N2 adsorption-desorption isotherm and pore property analysis.
    • Fig. S4. CeO2@ZIF-8 inhibits cell apoptosis and ROS overproduction induced by t-BOOH.
    • Fig. S5. Change in cell microstructure and mice body weight.
    • Fig. S6. Brain protection effects of CeO2@ZIF-8 in C57 MCAO mice.
    • Fig. S7. Fluorescence imaging and biodistribution analysis of CeO2@ZIF-8 in vivo.
    • Fig. S8. H&E staining of the heart, liver, spleen, lung, and kidney after treatment with CeO2@ZIF-8 for 3 days in MCAO mice model.
    • Fig. S9. Toxicity evaluation of CeO2@ZIF-8 in vivo.
    • Table S1. Pharmacokinetic parameters of CeO2 nanopolyhedra and CeO2@ZIF-8 composite nanomaterials.

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