Simultaneous Blood Brain Barrier Crossing and Protection for Stroke Treatment Based on Edaravone-Loaded Ceria Nanoparticles
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1 Supporting Information Simultaneous Blood Brain Barrier Crossing and Protection for Stroke Treatment Based on Edaravone-Loaded Ceria Nanoparticles Qunqun Bao, Ping Hu, * Yingying Xu, Tiansheng Cheng, Chenyang Wei, Limin Pan * and Jianlin Shi * State Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, , China. University of Chinese Academy of Sciences, Beijing, , PR China. School of Physical Science and Technology, ShanghaiTech University, Shanghai, , PR China. * addresses: jlshi@mail.sic.ac.cn (J. L. Shi), huping@mail.sic.ac.cn (P. Hu), lmpan@msn.cn (L. M. Pan)
2 Table 1: A glossary of abbreviations in this paper Full name reactive oxygen species blood brain barrier Angiopep-2 poly ethylene glycol low-density lipoprotein receptor-related protein the nanoplatform of ceria cores, organic ANG/PEG shell, and edaravone loaded in the shell superoxide dismutase catalase inductively coupled plasma optical emission spectrometry brain capillary endothelial cells confocal laser scanning microscopy rhodamine B isothiocyanate 5,5'-dimethylpyrroline-1-oxide methyl violet Cell Counting Kit-8 tert-butyl hydroperoxide 2,7-Dichlorodi-hydrofluorescein diacetate Evans Blue 4',6-diamidino-2-phenylindole fluorescein isothiocyanate propidium iodide Abbreviation ROS BBB ANG PEG LRP E-A/P-CeO 2 SOD CAT ICP-OES BCECs CLSM RITC DMPO MV CCK-8 tbhp DCFH-DA EB DAPI FITC PI
3 Figure S1. Various materials characterization to demonstrate successful preparation (a) FT-IR spectra of the oleylamine coated ceria (noted as OA-CeO 2 ), amine-poly (ethylene glycol)-carboxyl (NH 2 -PEG 1k -COOH) modified ceria (noted as P-CeO 2 ), Angiopep-2 (TFFYGGSRGKRNNFKTEEYC) and PEG co-decorated ceria nanoparticles (noted as A/P-CeO 2 ) for verifying the exchange of ligands. (b) Changes of hydrodynamic diameter distribution and Zeta potential of the as-prepared ceria nanoparticles. (c) UV-Vis absorbance spectra of solutions of free edaravone (noted as Edaravone) and the supernatant after centrifugation from E-A/P-CeO 2 solution (noted as Supernatant). (d) Standard curve of edaravone to determine the loading amount of edaravone in PEG/ANG-modified ceria nanoparticles (noted as E-A/P-CeO 2 ).
4 Figure S2. (a) UV-vis absorption spectra of MV (black line), the mixture of MV and Fenton agent (red line), the mixture a (solid curves) of E-A/P-CeO 2, Fenton agent and MV, and the mixture b (dashed curves) of A/P-CeO 2, Fenton agent and MV at varied E-A/P-CeO 2 concentrations for evaluating radical scavenging activity. All solutions had a total volume of 3 ml and an incubation time duration of 5 min in dark. (b) Reversible catalytic activity of E-A/P-CeO 2 tested by transmitting UV-Vis spectroscopy. After H 2 O 2 injection, the spectrum had a large shift to the right, and then gradually returned to its
5 original state in the time course. And (c) color of the mixture changed from faint yellow to aurantium, and then faded to light yellow gradually in the following 10 days. XPS analysis at the surface of 2 mm E-A/P-CeO 2 after reacting with (d) 10 mm H 2 O 2 and 20 mm H 2 O 2, and (e) Fenton reagent (2 mm H 2 O 2 with 0.3 mm FeSO 4 ) and (4 mm H 2 O 2 with 0.6 mm FeSO 4 ) Figure S3. Comparison of the protection capability of ceria nanoparticles against cell death induced by different doses of tbhp. Cell viability examined by CCK-8 assay after being treated with 25 ppm ceria nanoparticles (P-CeO 2, A/P-CeO 2, E-A/P-CeO 2 ).
6 Figure S4. Digital photograph (top) of representative TTC-stained brain tissue of stroke, in 24 h of E-A/P-CeO 2 -injection in different concentrations. Volume proportion (bottom) of the impaired part to the whole brain analyzed by Image-Pro Plus.
7 Figure S5. (a) Standard curve of EB and the absorbance of cerebral homogenate from each EB-injected group after respectively being treated by saline (noted as control), P-CeO 2, A/P-CeO 2, E-A/P-CeO 2 in stroke and the normal brain (noted as sham). (b) EB micrograms per gram of brain tissue calculated from the EB exudation. The data presented shows the control of stroke treated with saline, *P<0.05 vs. the normal (sham-treated), as well as *P<0.05 vs. the E-A/P-CeO 2 -treated.
8 Figure S6. Variations of blood index of rats from the control group of saline and E-A/P-CeO 2 -treated groups at different doses of 5 mg/kg, 10 mg/ kg, 20 mg/kg in 30 days of feeding. Various vital hematology markers and all the key biochemistry parameters fluctuated in the normal ranges, indicating that E-A/P-CeO 2 has negligible toxicity.
9 Figure S7. (a) Bio-distribution of Ce per gram of tissues in brain and main tissues (heart, liver, spleen, lung and kidney) of injection of 0.5 mg/kg E-A/P-CeO 2 by intravenous administration. (b) Amounts of Ce in collected faeces and urine samples at several time points from E-A/P-CeO 2 -injected rats at the dose of 10 mg/kg. (c) A small amount of urine was taken to detect whether there existed E-A/P-CeO 2 particles or not by TEM and EDS.
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