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.
Additional Files
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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