Bao 2018

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Article
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
ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01994 • Publication Date (Web): 22 Jun 2018
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Page 1 of 45 ACS Nano
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4 Simultaneous Blood–Brain Barrier Crossing and
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7 Protection for Stroke Treatment Based on
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Edaravone-Loaded Ceria Nanoparticles
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12 Qunqun Bao,†‡§ Ping Hu,†* Yingying Xu,†§ Tiansheng Cheng,†§ Chenyang Wei,† Limin
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Pan†* and Jianlin Shi†*
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18 State Key Laboratory of High Performance Ceramics and Superfine Microstructures,
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21 Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050, China.
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University of Chinese Academy of Sciences, Beijing, 100049, PR China.
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27 §
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School of Physical Science and Technology, ShanghaiTech University, Shanghai,
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30 201210, PR China.
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34 * Email addresses: jlshi@mail.sic.ac.cn (J. L. Shi), huping@mail.sic.ac.cn (P. Hu),
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36 lmpan@msn.cn (L. M. Pan)
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ACS Nano Page 2 of 45
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4 ABSTRACT: Cerebral vasculature and neuronal networks will be largely destroyed due
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7 to the oxidative damages by over-produced reactive oxygen species (ROS) during stroke,
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9 accompanied by the symptoms of ischemic injury and blood–brain barrier (BBB)
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12 disruption. Ceria nanoparticles, acting as an effective and recyclable ROS scavenger,
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have been showing high effectiveness in neuroprotection. However, the brain access of
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17 nanoparticles can only be achieved by targeting the damaged area of BBB, leading to the
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20 disrupted BBB unprotected and turbulence of microenvironment in brain. Nevertheless,
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22 the integrity of BBB will cause very limited accumulation of therapeutic nanoparticles in
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25 brain lesions. This dilemma is a great challenge in the development of efficient stroke
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nanotherapeutics. Herein, we have developed an effective stroke treatment agent based on
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30 monodisperse ceria nanoparticles, which are loaded with edaravone and modified with
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33 Angiopep-2 and poly ethylene glycol on their surface (E-A/P-CeO2). The as-designed
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35 E-A/P-CeO2 features highly effective BBB-crossing via receptor-mediated transcytosis to
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38 access brain tissues and synergistic elimination of ROS by both the loaded edaravone and
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ceria nanoparticles. Resultantly, the E-A/P-CeO2 with low toxicity and excellent
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43 hemo/histocompatibility, can be used to effectively treat strokes by great intracephalic
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46 uptake enhancement and in the meantime effective BBB protections, holding great
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48 potentials in the stroke therapy with much mitigated harmful side-effects and sequelae.
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51 KEYWORDS: stroke, blood–brain barrier, ceria nanoparticles, edaravone, reactive
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oxygen species
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4 Stroke is one of the most common causes of disability which ranks No. 5 among all
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7 causes of death.1 Currently, the treatments of ischemic stroke mainly include
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9 thrombolytic and neuroprotective therapies.2 Due to the highly limited therapeutic time
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12 window, only a few patients (2% − 7%) can benefit from effective thrombolytic therapy
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within 4.5 h post onset.3 Free radical scavengers, as a kind of important neuroprotective
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17 agents, can prevent neurons from being damaged in a chain reaction induced by reactive
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20 oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide anion (O2-•) and
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22 hydroxyl radical (•OH), which are some of the core pathological causes of ischemic
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25 stroke.4 Although the effective neuroprotection by edaravone has been proved, several
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critical problems remain, such as the ineffectiveness in crossing the blood–brain barrier
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30 (BBB), repetitive and over-high dosage, and severe side effects. The application of
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33 nanobiotechnology, as free radical scavengers, provides promising approaches to
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35 achieve efficient treatments of stroke.
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38 Ultrasmall ceria nanoparticles can easily lose oxygen and/or electrons in the fluorite
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lattice structure, which leads to the generation of oxygen vacancies and lowered valence
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43 states of cerium atoms, and the resultant antioxidant activity based on electron transfer
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46 between cerium (III) and cerium (IV) (Scheme 1a). Besides, due to the memory function
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48 of the lattice structure and electron exchange with other ions, ceria nanoparticles will
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51 easily recover their redox activity, allowing repetitive elimination of ROS.13-16 As a result,
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ceria nanoparticles can be applied to mimic antioxidant enzymes (superoxide dismutase17
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56 (SOD) and catalase18 (CAT)) in the treatments of strokes associated with high oxidative
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4 stresses. However, a number of shortcomings still remain to be overcome: (i) To ensure
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7 the high biomimetic enzyme activity,19 ceria nanoparticles of smaller than 5 nm in
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9 diameter are more favorable. However, the ultrasmall sizes will lead to over-short
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12 vascular circulation half-life, and the easy inter-particle agglomeration of these ultrasmall
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ceria nanoparticles makes the issue much worse; (ii) Reluctant responses in capturing free
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17 radicals of extremely short half-life and free path20,21 due to the over-thick
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20 surface-modification of ceria nanoparticles for maintaining biocompatibility and
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22 stabilization in physiological environment; (iii) Most importantly, the potential BBB
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25 disruption for the sake of effective nanoparticle accumulation in brains. As the exclusive
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biological barrier, BBB protects the brain from potentially harmful compounds in the
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30 blood, but also unfortunately prevents the intracephalic accumulation of nanoparticles.22
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33 Since the BBB could be broken down in brain ischemia,23-25 only a tiny proportion of
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35 nanoparticles get access to brain lesion by targeting the damaged BBB area. 26 Thus the
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38 therapeutic efficacy for neuroprotection is still greatly limited. And the slow opening of
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the damaged BBB took several hours, resulting in the extremely low accumulation of
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43 nanoparticles in the brain lesion.27,28 More unfortunately, the stroke-induced disruptions
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46 of BBB will most likely cause neurological dysfunctions,24 such as acute cerebral edema
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48 and hemorrhage. Therefore, developing an elaborate strategy to achieve simultaneous
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51 BBB-crossing and protection for stroke treatment, is of great significance.
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In this study, a kind of BBB-targeting edaravone-loaded ceria nanoparticles has been
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56 first developed for simultaneous BBB-crossing and stroke treatment. As illustrated in
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4 Scheme 1, the designed nanoplatform exhibit a core-shell structure with monodispersed
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7 ultrasmall ceria as core, organic Angiopep-2 (ANG)/ poly ethylene glycol (PEG) as shell
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9 and edaravone loaded in the organic shell. It consists of three key components: (i) PEG
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12 significantly improves the biocompatibility as well as monodispersity, and extends the
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half-lives in the bloodstream.29 (ii) Edaravone loaded in the shell synergistically
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17 scavenges ROS, especially those not close to ceria core. Thus ROS scavenging efficiency
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20 was significantly improved. (iii) ANG as the targeting ligand on ceria nanoparticles,
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22 which was used to specially bind to the low density lipoprotein receptor-related protein
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25 (LRP) overexpressed on cells that comprise the BBB.30,31
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On the whole, the BBB-targeting nanoplatform, based on the ceria nanoparticle core/
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30 PEG-ANG shell structure, is able to competently cross BBB via ANG-LRP-mediated
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33 transcytosis. During this process, BBB will be protected from oxidative stress-induced
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35 disruption in stroke owing to the synergistic elimination of ROS by ceria nanoparticle
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38 core and molecular edaravone absorbed in the organic shell. Moreover, in the brain lesion
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tissues beyond BBB where the nanoplatform has finally reached, ceria cores show highly
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43 sustained ROS scavenging capability in addition to the ROS-scavenging effect of
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46 edaravone retained in the nanoplatform, thanks to the reproducibility of cerium.
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48 Altogether, this work indicates that the nanoplatform based on ANG-conjugated and
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51 edaravone-loaded ceria nanoparticles can not only enhance the intracephalic uptake but
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also prevent the injuries on both brain tissues and BBB in stroke.
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RESULTS AND DISCUSSION
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6 Synthesis and Characterization of E-A/P-CeO2. Scheme 1b illustrates the structure
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9 of E-A/P-CeO2 nanoparticles, which were synthesized according to the following main
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steps. First, ceria nanoparticles with oleylamine on the surface (OA-CeO2) were obtained
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14 by a catalytic pyrolysis method.32 Subsequently, owing to the strong ligand binding
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17 between ceria and carboxyl/thiol groups,30,33 the ceria nanoparticles were decorated with
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19 ANG and amine poly (ethyleneglycol)-carboxyl (NH2-PEG1k-COOH) in parallel
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22 (A/P-CeO2) through ligand exchange with oleylamine. Finally, the molecular edaravone
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was loaded in the organic layer on the ceria nanoparticles (E-A/P-CeO2) by van der Waals
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27 forces and organic macromolecules absorption. As shown in the transmission electron
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30 microscopic (TEM) images (Figure 1a), OA-CeO2 in hexamethylene are uniform and
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32 near-spherical with an average diameter of 4.3 ± 0.5 nm. After coated with ANG/PEG, the
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35 ceria nanocrystals were transferred from oleic phase into aqueous solutions (Figure 1b),
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which resulted in excellent dispersibility and quasi-spherical morphology. To verify the
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40 successful exchange of ligands, Fourier transform infrared spectroscopy (FT-IR) and
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43 Zeta-potential measurements were carried out (Figure S1a, b). The FT-IR spectra show
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45 the disappearance of carbon-carbon double bonds of oleylamine along with the
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48 appearance of ether linkages of PEG and reinforcement of carbonyl groups of ANG,
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indicating that PEG and ANG have replaced oleylamine as the coating layer on ceria
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53 nanoparticles. These results were further confirmed by the decreased potential by
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56 Zeta-potential analysis. And a peak corresponding to sulfur (S) belonging to ANG can be
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4 observed from the Energy-dispersive X-ray spectrum (TEM/EDS) that further verifies the
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7 successful conjugation of the ANG chains (Figure 1c). Moreover, the ceria nanoparticles
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9 after surface modification still possess a pure and typical fluorite cubic structure as
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12 evidenced by X-ray diffraction (XRD) analysis (Figure 1d). To demonstrate that the final
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product of small ceria nanoparticles has a higher percentage of cerium (III), the surface
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17 amounts of cerium (III) oxide (peaks at 885.0 and 903.5 eV) and cerium (IV) oxide
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20 (peaks at 882.1, 888.1, 898.0, 900.9, 906.4, and 916.4 eV) were quantified by X-ray
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22 photoelectron spectroscopy (XPS). The results confirm that the slightly yellow ceria
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25 nanoparticles have a cerium (III) oxide content of ∼ 34.1% (Figure 1e), which ensures
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that the ceria nanoparticles possess antioxidant activity so as to mimic superoxide SOD
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30 and CAT.
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33 The diameters of PEG/ANG modified ceria nanoparticles (A/P-CeO2) based on
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35 dynamic light scattering measurements increased to 19.17 ± 5.24 nm (Figure S1b) due to
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38 the presence of hydrated layers, which may prevent ROS from reaching the ceria
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nanoparticle cores directly and then reduce the ROS-scavenging activity because of the
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43 very short effective half-life and limited free path of ROS.20,21 To capture those ROS not
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46 reaching the core of ceria nanoparticles, edaravone, serving as molecular ROS scavenger,
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48 has been absorbed in the organic macromolecule layer (ANG/PEG) by molecular
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51 interaction with A/P-CeO2 during stirring for 12 h, and the final product is named as
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E-A/P-CeO2. After centrifugation, the supernatant and initial edaravone solutions were
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56 both diluted ten-folds and measured by UV-Vis absorption spectroscopy (Figure S1c).
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4 Edaravone loading was quantified by an internal standard method based on the standard
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7 curve at the edaravone characteristic peak (λ = 239 nm) (Figure S1d). And the optimized
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9 loading amount of edaravone in A/P-CeO2 was 0.083 g/g at the edaravone to A/P-CeO2
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12 concentration ratio of 1.125 : 1. Then, the release profile of the edaravone in E-A/P-CeO2
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nanoparticles has been obtained in Figure 1f, which shows very negligible edaravone
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17 release (lower than 4% in 24 h) in the physiological condition. The result indicates that
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20 most edaravone will not leak out significantly. This means that edaravone molecules
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22 loaded in E-A/P-CeO2 will be mostly retained in an intact nanoparticle rather than
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25 detached from the E-A/P-CeO2.
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Enhanced Cellular Uptake and BBB-Crossing in Vitro. To evaluate whether the
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30 as-synthesized E-A/P-CeO2 have acquired high enough targeting capability in vitro, the
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33 cellular uptake of E-A/P-CeO2 by brain capillary endothelial cells (noted as BCECs), the
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35 main component of BBB, were intensively investigated. Due to the overexpress of LRP
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38 on cellular membrane, greatly enhanced cellular uptake of the E-A/P-CeO2 in comparison
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with P-CeO2 was confirmed by both flow cytometry and confocal microscopy. As shown
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43 in flow cytometric measurements (Figure 2a), BCECs treated with fluorescent dye
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46 Rhodamine B isothiocyanate (RITC) labelled E-A/P-CeO2 show 2.1 ~ 3.6 times higher
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48 positive fluorescence signal ratio than those treated with equimolar amounts of RITC
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51 labelled P-CeO2 after their incubation for the same time period, especially during first 1 h,
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indicating much more rapid E-A/P-CeO2 uptake by BCECs than P-CeO2. In addition,
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56 confocal microscopic analysis of BCECs by co-culture with these nanoparticles also
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4 presents the highly consistent results with the flow cytometry (Figure 2b). Strong red
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7 fluorescence signal can be observed around the nucleus of the BCECs when treated with
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9 E-A/P-CeO2. In contrast, only much weaker fluorescence signal is visible with P-CeO2
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12 under the same conditions. These data clearly confirm the significantly stronger cellular
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uptake of E-A/P-CeO2 than P-CeO2 by BCECs, both in quantity and rate, which can be
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17 attributed to the specific targeting effect of ANG ligands to the LRP receptor on the
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20 membrane of BCECs. The E-A/P-CeO2 uptake reached a maximum in 4 h of incubation,
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22 ensuring that E-A/P-CeO2 particles can timely protect BCECs against oxidation-induced
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25 death in the BBB.
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According to the literature reports,30,31,34 transwell filters seeded with BCECs will form
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30 a compact layer after proliferating, which could then be used to measure the
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33 BBB-crossing efficacy of nanoparticles (Figure 2c). The value of Transendothelial
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35 Electrical Resistance (TEER) represents the model’s permeability to inorganic ions and is
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38 the most sensitive and traditional indicator of BBB function. The obtained TEER values
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are typically above 200 Ω cm2 during the experiments, which indicate that the BCECs
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43 monolayer is in integrity and therefore meets the requirements of a BBB model in
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46 vitro30,34. Then we analyzed the Ce contents on the apical side (AP), cellular monolayer
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48 (FIL), and basolateral side (BL) by inductively coupled plasma optical emission
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51 spectrometry (ICP-OES) after co-incubating in the apical medium with these
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nanoparticles over 24 h. Figure 2d reveals that 21.0 ± 2.4% of the targeting E-A/P-CeO2
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56 has penetrated across the BBB layer and been detected on the basolateral side. In
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4 comparison, only 6.2 ± 1.0% of non-targeting P-CeO2 was found under the same
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7 condition, indicating the significant targeting effect of E-A/P-CeO2 compared with
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9 P-CeO2. To make sure whether the ANG ligands on E-A/P-CeO2 had played a major role
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12 in mediating the BBB-crossing transcytosis with LRP receptors or not, typical blocking
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assay was further employed. High-dose free ANG was added into the apical medium 0.5
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17 h in advance to block the targeting interaction between E-A/P-CeO2 and LRP receptors.
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20 Resultantly the E-A/P-CeO2 amount on the basolateral side significantly decreased to 8.9
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22 ± 1.2%, similar to the level of non-targeting P-CeO2 nanoparticles, confirming the
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25 positive role of ANG on BBB targeting. These results clearly demonstrate the advantage
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of E-A/P-CeO2 in transporting across the BBB by endocytosis, resulting in the enhanced
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30 intracephalic uptake and guaranteeing the following neuroprotection effect.
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33 Regenerable ROS Scavenging and BCECs Protection in Vitro. The electron spin
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35 resonance (ESR) spectroscopy (Figure 3a) was used to detect the generated hydroxyl
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38 radicals in Fenton reaction by using 5,5'-dimethylpyrroline-1-oxide (DMPO) as the spin
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trapping agent. The appearance of 1:2:2:1 multiple peaks in the ESR spectrum evidences
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43 the presence of DMPO-OH adducts, suggesting the generation of hydroxyl radicals which
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46 is in good agreement with the literature report.35,36 Due to the competitive relations
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48 between DMPO and ceria nanoparticles in trapping radicals, the decrease of signal
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51 intensity will prove the hydroxyl radicals scavenging effect of ceria nanoparticles during
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the reaction in a mixture containing Fenton agent, ceria nanoparticles and DMPO.
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56 Interestingly, the amount of spin adduct sharply decreased upon adding E-A/P-CeO2
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4 rather than equal molar amounts of A/P-CeO2, confirming that edaravone loaded in
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7 A/P-CeO2 could efficiently promote the ROS scavenging. Similarly, the radical
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9 scavenging role of ceria nanoparticles was also examined by UV/Vis spectroscopy using
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12 methyl violet (MV)37 (Figure S2a). The maximum absorbance of MV (λ = 582 nm)
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largely decreased because of the carbon-carbon double bond breakage through the
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17 electrophilic addition by hydroxyl radical in Fenton reaction. However, the absorption
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20 decay of MV was not significant when free radical scavengers A/P-CeO2 (the mixture b)
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22 or E-A/P-CeO2 (the mixture a) were added. These results demonstrate that E-A/P-CeO2
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25 can adsorb and eliminate the hydroxyl radicals to protect the MV from oxidation.
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Meanwhile, the catalytic effect and regenerability of E-A/P-CeO2 during the reaction
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30 with H2O2 were evaluated by in-situ Raman spectroscopy excited by 488 nm laser. As
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33 shown in Figure 3b, the major peaks centered at 460 cm−1 can be indexed as a symmetric
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35 breathing mode of the oxygen atoms around Ce ions related to the ordering in the oxygen
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38 sublattice.38 After adding H2O2 into the above system, the major peaks immediately shift
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to 850 cm−1 and 880 cm−1 while the peak at 460 cm−1 disappears because of the reaction
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43 between ceria nanoparticles and H2O2, leading to the generation of O–O stretching of
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46 adsorbed peroxide species (O22−) accompanied by defect aggregation and disordering of
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48 oxygen sublattice. As the reaction proceeds, the peaks at 850 cm−1 and 880 cm−1 become
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51 gradually weaker, while the one at 460 cm−1 intensifies and approximately returns to the
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original intensity in 1 h. Here, the intensity changes of the 850 cm−1 and 880 cm−1 peaks
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56 from their generation back to disappearance are regarded as an anti-oxidation cycle. As
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4 anticipated, adding H2O2 again, the changes of the peak intensities in the second cycle are
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7 almost identical to the first cycle in the Raman spectra, indicating an excellent recyclable
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9 antioxidant property of E-A/P-CeO2. To further verify this, E-A/P-CeO2 reacted with
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12 H2O2 was also evaluated by transmitting UV-Vis spectroscopy.39 As shown in Figure S2b,
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after the injection of H2O2 into the aqueous solution of E-A/P-CeO2, transmitting UV-Vis
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17 absorption peak shifted to the right and the color gradually became darker, suggesting
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20 that cerium (III) on the E-A/P-CeO2 surface has been oxidized to cerium (IV). In the
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22 following 10 days, transmitting UV-Vis absorption peak of E-A/P-CeO2 reversible shifted
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25 to the left gradually, which implies the regeneration of Ce (III) on the surface of ceria
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nanoparticles. The results confirm the auto-regenerative and anti-oxidant properties of the
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30 E-A/P-CeO2. Furthermore, the amount of cerium (III) oxide on the surface was
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33 quantitatively analyzed by XPS. The results in Figure S2d and S2e show that the surface
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35 amounts of cerium (III) oxide decrease upon treating with ROS (H2O2 or •OH from
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38 Fenton reaction). Moreover, as the ROS concentration increases, the amount of the
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cerium (III) oxide also decreases. These results further indicate that E-A/P-CeO2 has an
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43 excellent capability of capturing and eliminating ROS. Thus, the above behavior indicates
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46 that E-A/P-CeO2 can be used as an effective biomimetic antioxidant such as SOD and
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48 CAT to eliminate ROS for stroke treatment.
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51 The antioxidant activity at cellular level was evaluated on BCECs. First of all, to rule
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out the materials' cytotoxicity, Cell Counting Kit-8 (CCK-8) assays were carried out
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56 (Figure 3c), which demonstrated the negligible cytotoxicity of these E-A/P-CeO2
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4 nanoparticles against BCECs in vitro. And then, to investigate the protective effect
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7 against oxidative stress-induced cell death, BCECs were treated with tert-butyl
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9 hydroperoxide (tBHP) that could trigger the generation of large amounts of intracellular
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12 ROS,26 and then tested using CCK-8 assay. As shown in Figure 3d, the cell viabilities
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have been largely reduced after co-incubation with the different doses of tBHP. Once
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17 protected by ceria nanoparticles against oxidative stress-mediated injury, the cell survival
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20 rate shows no noticeable decrease, but even slight increase at the low concentration (25
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22 ppm, Figure S3). To reveal the protective effects of different kinds of ceria-based
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25 nanoparticles, cells incubated with P-CeO2, A/P-CeO2, E-A/P-CeO2 nanoparticles were
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tested. The higher cell viability with A/P-CeO2 than that with P-CeO2 demonstrates the
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30 contribution of ANG-targeting in enhancing the cellular uptake, which efficiently and
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33 timely eliminates intracellular ROS. The highest cell viability by adding E-A/P-CeO2
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35 further indicates that edaravone loaded in A/P-CeO2 could synergistically elevate the
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38 ROS scavenging activity.
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For further quantitative analysis, Annexin V-fluorescein isothiocyanate (FITC)/
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43 propidium iodide (PI) Apoptosis Detection kit was used to measure the BCECs apoptosis
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46 ratio by flow cytometer (Figure 3e). It can be found that E-A/P-CeO2 shows the highest
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48 percentage of live cells (41.1%) and the lowest percentage of late apoptosis cells (25.0%)
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51 among the samples, in comparison to those of A/P-CeO2 (37.9% live and 31.5% late
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apoptosis) and P-CeO2 (19.7% live and 34.6% late apoptosis). All the above results
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56 demonstrate that E-A/P-CeO2 is capable of effectively protecting BCECs and BBB
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4 against ROS attack by efficiently eliminating intracellular ROS owing to the enhanced
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7 cellular uptake, and the synergistic ROS-scavenging functions by both loaded edaravone
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9 in the outer shell and the ceria core in the constructed nanoplatform, which could
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12 guarantee its further in vivo application in mitigating oxidative stress and preventing
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cerebral lesion.
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17 BBB-Crossing Transport and Protection against Stroke in Vivo. In order to
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20 demonstrate the BBB-crossing capability, cerebral uptake experiments of the ceria
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22 nanoparticles were carried out on healthy rats, whose BBB was complete to exclude the
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25 effect of damaged BBB in stroke. After intravenous administration, amounts of ceria
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nanoparticles in brain tissue were tested by ICP-OES (Figure 4b). Compared with
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30 P-CeO2, E-A/P-CeO2 shows multifold-enhanced accumulation in brain region in 24 h
31
32
33 after injection, indicating that E-A/P-CeO2 nanoparticles have been effectively delivered
34
35 across the BBB into the brain by an ANG targeting and LRP receptor mediated
36
37
38 transcytosis process in vivo. Furthermore the excellent monodispersity and ultrasmall size
39
40
also contribute to the cerebral accumulation of the nanoparticles. Meanwhile, the
41
42
43 bio-distribution of E-A/P-CeO2 in main tissues (heart, lung, kidney, spleen and liver)
44
45
46 were also observed (Figure S7a). Although a large majority of E-A/P-CeO2 has
47
48 accumulated in spleen and liver, the quantity of ceria nanoparticles in per gram brain
49
50
51 tissue is still much higher than the others. Such a largely enhanced intracerebral uptake
52
53
capability of the targeting E-A/P-CeO2 delivery system may offer promising
54
55
56 opportunities for in vivo cerebral disease therapy.
57
58
59
1
2
3
4 Furthermore, to explore the therapeutic effect of ceria nanoparticles in vivo, the focal
5
6
7 cerebral ischemic injury of rat was tested using middle cerebral artery occlusion (MCAO)
8
9 mode40 by tail vein injection. After stroked for 24 h, the cerebral infarct volume was
10
11
12 measured by triphenyltetrazolium chloride (TTC) staining, showing the stained normal
13
14
tissue in red and the unstained infarction in white. The result shows that the infarct
15
16
17 volume of the control group of saline injection is as high as 45.6 ± 4.8%. However, when
18
19
20 treated with varied doses of E-A/P-CeO2, the infarct volumes considerably decrease
21
22 down to 15.0 ± 4.1% at an optimal concentration of 0.6 mg/kg (Figure S4a,b). We further
23
24
25 compared the neuroprotective effects of P-CeO2, A/P-CeO2, and E-A/P-CeO2 at the same
26
27
injection dose of 0.6 mg/kg (Figure 4c). Owing to the enhanced accumulation in brain
28
29
30 tissue via ANG targeting, A/P-CeO2 shows more significant neuroprotective effect than
31
32
33 P-CeO2, and comparatively E-A/P-CeO2 performs the best, which can be ascribed to the
34
35 synergistic ROS-scavenging effects by the edaravone component loaded in the targeting
36
37
38 systems and the ceria core for the largely improved treatment effect in vivo. Meanwhile,
39
40
considering that the oxidative damage induced by ROS was the major cause of stroke
41
42
43 injury, fluorescent probe of 2,7-Dichlorodi–hydro fluorescein diacetate (DCFH-DA) was
44
45
46 used to detect ROS levels in the brain after ischemia induction for the further
47
48 investigation of antioxidant protective effect of E-A/P-CeO2. ROS in frozen brain
49
50
51 sections could oxidize non-fluorescent DCFH to produce red fluorescent DCF based on
52
53
confocal configuration detection. As shown in the image (Figure 4d), the ROS level in the
54
55
56 E-A/P-CeO2 nanoparticles injected brain is markedly reduced compared to the stroke
57
58
59
1
2
3
4 control, indicating the highly efficient ROS elimination by E-A/P-CeO2. All the above in
5
6
7 vivo results demonstrate that E-A/P-CeO2 are capable of efficient accumulation in brain
8
9 tissue by BBB-crossing instead of BBB damaging, which is expected to resultantly
10
11
12 enhance the therapeutic efficacy of stroke by mitigating the intracerebral oxidative stress.
13
14
Prevention of the BBB Damage in Vivo. According to the previous reports,24,25
15
16
17 ischemia-hypoxia in stroke would result in cell apoptosis, increased endothelial cell
18
19
20 permeability and disassembly of tight proteins and intercellular junctions, which
21
22 consequently lead to BBB disruption. As a natural defense system to maintain the
23
24
25 stability of the brain micro-environment, the damage of BBB is reversible during early
26
27
stage but will become permanent inflammatory several days later, which might cause the
28
29
30 more severe lesions such as vasogenic cerebral edema and hemorrhagic transformation.
31
32
33 Thus, it is highly important to protect the BBB integrity in the ischemic stroke treatment.
34
35 It has been found in this study that the as-synthesized E-A/P-CeO2 features high
36
37
38 BBB-crossing capability via receptor-mediated (ANG-LRP) endocytosis, during which
39
40
part of the E-A/P-CeO2 nanoparticles may remain in BCECs to eliminate the oxidative
41
42
43 stress in BBB induced by ischemia-hypoxia as discussed above, and thus BBB integrity
44
45
46 can be protected. To verify this, the traditional Evans Blue (EB) staining assay,41 in which
47
48 EB cannot penetrate through the complete BBB to stain brain tissue, was performed to
49
50
51 test the permeability of BBB by measuring the amount of extravasated EB. As shown in
52
53
the digital photographs (Figure 5a), compared with normal rat (sham), the right half of
54
55
56 the brain in control group presents bright blue color after ischemic injury, demonstrating
57
58
59
1
2
3
4 that EB has clearly extravasated across the BBB into brain tissue. All ceria
5
6
7 nanoparticles-treated groups (P-CeO2, A/P-CeO2, E-A/P-CeO2) show much less
8
9 significant EB staining in the right halves than the control of stroke. More importantly,
10
11
12 the brain EB staining of E-A/P-CeO2-treated rat is the slightest. In accordance with the
13
14
results of EB fluorescent signal detection in brain slices by confocal microscopy (Figure
15
16
17 5b), the brain tissue of E-A/P-CeO2-treated rat shows the weakest red fluorescence of EB.
18
19
20 Quantitative analysis (Figure S5a) of EB exudation from blood into the brain by
21
22 spectrophotometer (Figure S5b) shows that the amount of EB uptake in brain of
23
24
25 E-A/P-CeO2-treated rats is the lowest. The above data clearly indicate that the
26
27
E-A/P-CeO2-treated rats have maintained the highest level of BBB integrity among all
28
29
30 groups tested, demonstrating that E-A/P-CeO2 featuring the ANG-targeting effect can
31
32
33 effectively accumulate within the cerebral microvascular endothelial cells and eliminate
34
35 ROS therein to prevent BBB from breakdown under the attack by oxidase-generated
36
37
38 superabundant ROS.42, 43
39
40
The long-Term Toxicity of E-A/P-CeO2 in Vivo. Although ceria nanoparticles,
41
42
43 possessing a wide range of potential biomedical applications, have attracted extensive
44
45
46 interests, however, its potential toxicity in treatment of stroke is still unclear to date16.
47
48 Herein, toxicity studies using Sprague-Dawley rats were performed. The rats after
49
50
51 intravenous injections of E-A/P-CeO2 nanoparticles in different dosage groups (0, 5, 10,
52
53
20 mg/kg) for 30 days were euthanized. The blood was acquired by abdominal aortic
54
55
56 method for biochemistry and hematology tests, while hematoxylin and eosin (H&E)
57
58
59
1
2
3
4 stained main organs for histological analysis. The H&E staining results of major organs
5
6
7 (heart, liver, spleen, lung, and kidney) show no significant acute, chronic pathological
8
9 toxicity, and adverse effects among the control group and the treatment groups (Figure
10
11
12 5c). A series of blood indexes including key biochemistry parameters and organs function,
13
14
in the drug treatment groups show no abnormities compared to those of the control group
15
16
17 (Figure S6). These results preliminarily prove that the E-A/P-CeO2 exhibits no significant
18
19
20 appreciable toxicity in vivo.
21
22 Besides, effective clearance of these nanoparticles from the body is another key factor
23
24
25 for biosafety requirements in vivo. To investigate the metabolic pathway of E-A/P-CeO2,
26
27
feces and urine of the treated rats were collected for an entire week and quantitatively
28
29
30 detected by ICP-OES. As shown in Figure S7a-b, only a small fraction of excretion was
31
32
33 through kidney into urine, which was further confirmed by TEM and corresponding EDS
34
35 (Figure S7c) examinations of a random urine sample. The vast majority of E-A/P-CeO2
36
37
38 was excreted via feces due to the larger hydrodynamic diameter than the renal filtration
39
40
threshold of 5.5 nm. About 7.0% and 49.6% of the E-A/P-CeO2 nanoparticles were
41
42
43 cleared by urine and feces within a week, respectively, which exhibit that these
44
45
46 nanoparticles could be well excreted from the body to ensure the high biosafety.
47
48
49
50 CONCLUSIONS
51
52
53 In summary, a promising neuroprotective nanoplatform, E-A/P-CeO2 composed of
54
55
56 monodispersed ultrasmall ceria cores, organic PEG and ANG shell and edaravone loaded
57
58
59
1
2
3
4 in the shell, has been successfully constructed and evaluated. It has been demonstrated
5
6
7 that such a nanoplatform is capable of effective BBB crossing thanks to the specific
8
9 interaction between ANG peptide and LRP receptor. Consequently, BBB will be
10
11
12 protected against the ROS attack due to synergistic ROS scavenging by both loaded
13
14
edaravone in the outer shell and the ceria core. In the meantime, the timely treatment of
15
16
17 ROS-induced brain injury in stroke will be achieved by extravasated nanoplatforms. On
18
19
20 one hand, E-A/P-CeO2 is a targeting nanodrug capable of active BBB crossing via
21
22 receptor-mediated transcytosis instead of passive diffusion through the damaged area of
23
24
25 BBB for stroke treatment. On the other hand, it is also an effective antioxidant agent for
26
27
preventing BBB against breakdown in stroke, providing a promising strategy to
28
29
30 overcome the contradiction between maintaining BBB integrity to ensure the brain
31
32
33 micro-environment stability and the efficient nanoparticle extravasation across BBB for
34
35 highly accumulation in intracerebral lesions. Meanwhile, low toxicity and good
36
37
38 hemo/histocompatibility of E-A/P-CeO2 have also been demonstrated. Therefore, this
39
40
work highlights an efficient strategy for the targeted treatment for brain nerve lesions and
41
42
43 the concurrent BBB protection against damages caused by stroke as well, and may shed a
44
45
46 light on other neurodegenerative disease treatments.
47
48
49
50 METHODS
51
52
53 Materials. Cerium (III) nitrate hexahydrate (Ce(NO3)3·6H2O, 99%), oleylamin (OA,
54
55
56 70%), 1-octadecene (ODE, 90%), cyclohexane (99.5%), and
57
58
59
1
2
3
4 5,5-dimethyl-1-pyrroline-N-oxide (DMPO, 98%) were purchased from Sigma-Aldrich.
5
6
7 NH2-PEG1k-COOH were acquired from Jenkem Co, Ltd. Angiopep-2
8
9 (TFFYGGSRGKRNNFKTEEYC) was synthesized by Chinese Peptide Company.
10
11
12 Edaravone was purchased from J&K scientific. Methyl violet (MV), hydrogen peroxide
13
14
(H2O2, 30%), Ferrous sulfate heptahydrate (FeSO4·7H2O, 99%) were obtained from
15
16
17 Sinopharm Chemical Reagent Co, Ltd.
18
19
20 Synthesis of Ceria Nanoparticles.32 First, ceria nanoparticles from CeO2-Oleylamine
21
22 were prepared through the reported method. Ce(NO3)3•6H2O as the biocompatible
23
24
25 precursor44,45 (0.43 g, 1.0 mmol) and oleylamine (0.802 g, 3.0 mmol) were added into
26
27
1-octadecene (ODE 4 g). The mixed solution was gently stirred for 1 h at room
28
29
30 temperature, and then put at 80 °C for 2 h in an argon atmosphere. Following heated and
31
32
33 maintained at 260 °C for 1.5 h. After cooled, the yellow precipitated ceria nanoparticles
34
35 dissolved in 5 ml hexamethylene and added ethanol or acetone using centrifugation
36
37
38 (20,000 rpm for 20 min) for three times. The resulting nanoparticles were dispersible in
39
40
20 ml hexamethylene. Subsequently, the ceria were decorated with Angiopep-2
41
42
43 (TFFYGGSRGKRNNFKTEEYC) and aminepoly-(ethylene glycol)-carboxyl
44
45
46 (NH2-PEG1k-COOH) through ultrasound. ANG (4 mg) and PEG (15 mg) were dissolved
47
48 in 10 mL of ultrapure water. Next 1 mL of ceria nanoparticles/ethyl ether solution (3
49
50
51 mg/mL) was added, which resulted in the ANG/PEG substitution for oleylamine ligand,
52
53
i.e., hydrophobic ceria being transfered into aqueous phase, under the ultrasonic
54
55
56 treatment. A clear faint yellow solution (A/P-CeO2/water) was obtained by stirring at
57
58
59
1
2
3
4 room temperature for 24 h to evaporate the methanol. The ceria nanoparticles were
5
6
7 collected by ultra-centrifugation and washed with deionized water several times to
8
9 remove excess ANG and PEG. And then, purified A/P-CeO2 nanoparticles were dispersed
10
11
12 in deionized water and calculated the molality at 1.6 mM by ICP-OES. Edaravone (5 mL)
13
14
with different concentration (0.6, 1.2, 1.8, 2.4 mM) was respectively added to 5 mL of
15
16
17 A/P-CeO2/water, and the resulting solution was stirred for 12 h. The mixture and the
18
19
20 supernatant by ultra-centrifuged (20,000 rpm for 120 min) were collected and tested by
21
22 UV-vis, respectively. The loading efficiency of edaravone from E-A/P-CeO2 were
23
24
25 calculated as follows: (Wini-Wsup)×100%/WCe (Wini: initial amount of edaravone and
26
27
Wsup: supernatent amount of edaravone).
28
29
30 For assessing the drug release behavior, E-A/P-CeO2 nanoparticles were encapsulated
31
32
33 in a dialysis bag of cutoff molecular weight at 3500, and placed into 30 ml releasing
34
35 medium (pH = 7.4), which were performed on the 37 °C shaking table at shaking speed
36
37
38 of 100 rmp. At certain time intervals, the 3 mL releasing medium was taken out and
39
40
tested by UV-vis at 239 nm to get the released edaravone concentration, then poured back
41
42
43 into the medium. The drug-releasing percentage is calculated as follows: Wrel ×
44
45
46 100%/Wini (Wrel: the released amount of edaravone, Wini: the initial amount of
47
48 edaravone).
49
50
51 Characterization. Transmission electron microscope (TEM) and energy dispersive
52
53
X-ray spectroscopy (EDS) on JEM-2100F electron microscope were used to detect
54
55
56 morphology and elemental distribution analysis with a voltage of 200 kV. The X-ray
57
58
59
1
2
3
4 diffraction (XRD) was recorded by a Rigaku D/MAX-2250 V diffractometer with a Cu
5
6
7 Kα radiation target (40 kV, 40 mA). X-ray photoelection spectroscopy (XPS) on a
8
9 VGMicro MK II instrument was operated with monochromatic Mg Ka X-rays (150 W,
10
11
12 1253.6 eV). FTIR spectra on an IRPRESTIGE-21 spec-trometer (Shimadzu) were
13
14
measured in the attenuated total reflectance methods. UV–vis absorption and
15
16
17 transmission spectra were recorded on UV-3101PC Shimadzu spectroscope. Zeta
18
19
20 potential and hydrodynamic size were tested by Dynamic light scattering (DLS Malvern
21
22 ZetasizerNanoseries ZS90). Inductively coupled plasma optical emission spectrometry
23
24
25 (ICP-OES, Agilent 725, Agilent Technologies, US) was used to analyze Ce
26
27
concentrations of samples. The confocal microscopy (CLSM) images were recorded on
28
29
30 FV1000, Olympus, Japan. The flow cytometry (BD LSRFortessa) was utilized to analyze
31
32
33 cellular uptake and apoptosis.
34
35 Enhanced Cellular Uptake in Vitro. 5 mg ceria nanoparticles (P-CeO2, A/P-CeO2,
36
37
38 E-A/P-CeO2) were respectively mixed with 0.5 mg RITC in 5 ml water and the solutions
39
40
were stirred for 12 h. After ultracentrifugation, RITC-conjugated ceria nanoparticles were
41
42
43 dispersed in DMEM at a concentration of 50 ppm. BCECs were seeded into 6-well
44
45
46 microplates with a density of 105/well and allowed to adhere overnight at 37 °C under 5%
47
48 CO2. And then the culture medium was changed with as-prepared DMEM containing
49
50
51 ceria nanoparticles. After co-incubation for 1, 4 or 7 h, the BCECs were rinsed with PBS
52
53
three times to remove the non-uptaken ceria nanoparticles, and tested by a flow
54
55
56 cytometry analysis in RPhycoerythrin. Meanwhile, BCECs were seeded into a
57
58
59
1
2
3
4 CLSM-special cell culture dish and treated by the process described above, and then
5
6
7 stained with DAPI. Cellular uptake of ceria nanoparticles was detected by the confocal
8
9 fluorescence on an Olympus FV1000 laser-scanning microscope under the excitation at
10
11
12 358 nm for DAPI and 540 nm for RITC.
13
14
BBB-Crossing Model in Vitro. The BBB model was constructed by polycarbonate
15
16
17 24-well Transwell membrane of 1.0 mm mean pore size, 0.33 cm2 surface areas
18
19
20 (FALCON Cell Culture Insert, Becton Dickinson Labware, USA), in which BCECs were
21
22 seeded at a density of 104 cells/well and cultured for 4 days. Epithelial voltohmmeter
23
24
25 (Millicell-RES, Millipore, USA) was used to measure the transendothelial electrical
26
27
resistance (TEER) of cell monolayers, and those of above 200 Ω cm2 were selected for
28
29
30 experiments. The ceria nanoparticles (P-CeO2, E-A/P-CeO2 particles) diluted in Roswell
31
32
33 Park Memorial Institute medium (RPMI) 1640 at the concentration of 50 ppm were
34
35 added into the apical side chamber (imitating blood side in vivo) with 50 rpm shaking at
36
37
38 37 °C for 18 h. Finally, the apical side chamber, the filter membrane and the basolateral
39
40
medium were collected and analyzed of the Ce contents by ICP-OES. To further evidence
41
42
43 the transcytosis mechanism by ANG-mediated, a blocking study was carried out in
44
45
46 parallel by adding free ANG at a concentration of 3 mg/mL to the apical side chamber 0.5
47
48 h before adding E-A/P-CeO2 particles, followed by the identical above-mentioned steps.
49
50
51 Evaluation of the Antioxidation and Reproducibility in Vitro. ROS detection by
52
53
electron spin resonance (ESR) spectroscopy: 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)
54
55
56 was selected as the spin trapping agent (5 µL, 98%) in ESR spectrum (Bruker EMX1598
57
58
59
1
2
3
4 spectrometer) for evaluating the effect of ceria nanoparticles (100 µL, 200 ppm) in
5
6
7 scavenging free radical that produced in Fenton reaction (FeSO4: 70 µL, 0.735mM and
8
9 H2O2: 25 µL, 0.315 mM) in the sample of 200 µL of the total volume. The effects of
10
11
12 hydroxyl radicals elimination were related with the change of relative peak intensity of
13
14
the ESR spectra of DMPO-OH·adducts.
15
16
17 ROS detection by UV-vis spectroscopy: hydroxyl radical scavenging capability of ceria
18
19
20 nanoparticles was further detected by a photometric method in a system containing
21
22 methyl violet (MV) and Fenton agent. The sample containing 0.012 mM MV, 0.15 mM
23
24
25 FeSO4, 1.0 M H2O2, and an appropriate amount of ceria nanoparticles in phosphate buffer
26
27
solution (PBS, pH = 7.4) of 5 mL of total volume. After incubation in dark for 5 min, the
28
29
30 ultraviolet absorption of the reaction solutions was measured, and the antioxidation
31
32
33 performance was evaluated by comparing the maximum absorbance of MV in the
34
35 conditions with and without ceria nanoparticles.
36
37
38 Detection by Raman spectra: the sample solution containing 10 mg ceria nanoparticles
39
40
and 10 mg H2O2 in 0.1 mL distilled water was dropped on the glass sheet and measured
41
42
43 by Laser Raman Microscope (Thermo) under 488 nm laser excitation. The Raman spectra
44
45
46 of the as-prepared sample were obtained in the time course (0, 10, 30, 60 min).
47
48 Immediately after the first cycle, 10 mg H2O2 in 0.1 mL distilled water was dropped on
49
50
51 the previous sample again and the examination process was the same as the above.
52
53
Cytotoxicity Assessment in Vitro. Brain capillary endothelial cells (noted as BCECs,
54
55
56 Cellbio (Shanghai) Ltd) were maintained at 37 °C and with 5% CO2 in Dulbecco’s
57
58
59
1
2
3
4 Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), 1%
5
6
7 penicillin/streptomycin in a humidified incubator. Cells were generally plated in cell
8
9 culture flask (Corning, USA) and allowed to adhere for 24 h then harvested by treatment
10
11
12 with 0.25% trypsin-EDTA solution (Gibco, USA). BCECs viability was evaluated by a
13
14
standard CCK-8 assay (Cell Counting Kit, Beyotime Institute of Biotechnology, Shanghai,
15
16
17 China), which were seeded into 96-well tissue culture plates at the density of 106/well
18
19
20 and then incubated for 24 h at 37 °C and 5% CO2 to ensure the cell attach. Afterwards,
21
22 The culture medium was changed with fresh medium containing ceria nanoparticles at
23
24
25 different Ce concentrations (0, 3.125, 6.25, 12.5, 25, 50, 100, 200 ppm), In 24 h or 48 h,
26
27
the original medium was removed, CCK-8 solutions were added to each well of the
28
29
30 microtiter plate, incubated for another 1 h in a CO2 incubator, the absorbance of formazan
31
32
33 in each well was measured by an enzyme-linked immunosorbent assay reader (BioTeck
34
35 instrument, USA) at 490 nm to calculate the cell viability to the control group.
36
37
38 Protection against Intracellular ROS in Vitro. BCECs were grown in 96-well culture
39
40
medium plates at the density of 106/well for 24 h at 37 °C and 5% CO2, and then the
41
42
43 solution of tert-butyl hydroperoxide (tBHP) as an oxidant was added into each well. In
44
45
46 1.5 h, BCECs were washed with PBS three times and new media containing P-CeO2,
47
48 A/P-CeO2, or E-A/P-CeO2 (25 or 50 ppm) were added into to each well. In 6 h after the
49
50
51 treatments, standard CCK-8 assay was performed for viability tests. Meanwhile, BCECs
52
53
were also seeded into 6-well microplates at a density of 105/well and treated by the steps
54
55
56 described above. Finally, after co-incubation with ceria nanoparticles for 6 h, the BCECs
57
58
59
1
2
3
4 were immediately resuspended in 500 µl of binding buffer and stained by 2 µM Annexin
5
6
7 V-FITC for 15min, 4.5 µM propidium iodide (PI) for 10 min in the dark (Annexin
8
9 V-FITC/PI Apoptosis Detection Kit, Beyotime). the cell apoptosis was examined using
10
11
12 flow cytometry (BD LSRFortessa).
13
14
Transport across BBB and Distribution of Ceria Nanoparticles in Vivo. Healthy
15
16
17 Sprague-Dawley rats (about 220 g in weight) were purchased from Shanghai SLAC
18
19
20 Laboratory Animal Co.,Ltd. And all animal studies were based on the guidelines by the
21
22 Institutional Animal Care and Use Committee.
23
24
25 P-CeO2 and E-A/P-CeO2 were respectively dispersed in saline solution after ultraviolet
26
27
disinfection, and then were injected into the two groups of rats by the tail vein with a
28
29
30 dose of 0.5 mg/kg. After 24 h, the brains, hearts, kidneys, livers, spleens, lungs were
31
32
33 rapidly acquired and weighed up after decapitating the rats. The samples of tissues
34
35 dissolving in aqua regia for one month were obtained to measure the concentrations of Ce
36
37
38 by using ICP-OES.
39
40
41
Protection against Stroke in Vivo. According to the previous reports, ischemic stroke
42
43
44 was successfully induced by constructing Middle Cerebral Artery Occlusion (MCAO)
45
46
47 Model. Subsequently, E-A/P-CeO2 particles dispersed in saline solution were
48
49 intravenously injected into rats at varied doses of 0, 0.3, 0.6, 0.9 mg/kg. In 24 h, these
50
51
52 rats were perfused with saline, then, their brains were quickly harvested and stained by
53
54
2,3,5-triphenyltetrazolium chloride (TTC). Finally, these brains were cut into five slices
55
56
57
58
59
1
2
3
4 after immediate freezing, and photos were taken using digital camera. The normal tissues
5
6
7 were stained red while the ischemia tissues did not produce change as shown pale. The
8
9 percentage of infarct volumes were analyzed by Image-Pro Plus. To compare the effects
10
11
12 among P-CeO2, A/P-CeO2, E-A/P-CeO2, these nanoparticles were respectively
13
14
intravenously injected into different rats by the above process at the same dose.
15
16
17 To further testify the effect of ceria nanoparticles on inhibiting cell apoptosis in vivo,
18
19
20 Reactive Oxygen Species Assay Kit (ROS assay kit based on DCFH-DA) was used to
21
22 mark the brain area with DAPI being used as a counter stain in nucleus, and then kept at
23
24
25 37 °C for 30 min in dark place. By Fluorescent Microscopy test, DAPI-stained nuclei
26
27
became blue (emission monitored at 420 nm) under UV excitation at 330-380 nm, and
28
29
30 ROS positive cells labeled by fluorescein were red (emission monitored at 590 nm) under
31
32
33 excitation at 510-560 nm.
34
35 Prevention of the BBB Damage in Vivo. The rats were divided into five groups. One
36
37
38 group was healthy as sham-operated group, others were quickly treated with saline,
39
40
P-CeO2, A/P-CeO2 and E-A/P-CeO2 particles at the same dose in 22 h of stroke. Then
41
42
43 each group was subject to the following steps of experiments. Briefly, in 22 h of stroke,
44
45
46 two percent EB was dissolved in saline and injected into rats as a BBB permeability
47
48 tracer at the dose of 4 ml/kg. After circulation for 2 h, the rat chest walls were opened to
49
50
51 perfuse with saline through the left ventricle. The brains were carefully removed and
52
53
weighed up, then, were taken photos using a digital camera. Meanwhile, the brain tissue
54
55
56 was frozen and sliced of around 10 µm in thickness, and the permeability of EB was
57
58
59
1
2
3
4 observed at 680 nm under a confocal laser scanning microscope under the excitation at
5
6
7 620 nm. To further quantify the amount of EB, the brain was cut into small fragments and
8
9 incubated in dimethyl formamide for 24 h at 60 °C. The homogenate of the brain was
10
11
12 centrifuged and the supernatant was taken out for absorbance measurement at 620 nm by
13
14
the microplate reader, then, the content of the extraction of EB was calculated via
15
16
17 standard concentration line.
18
19
20 Toxicity Assay and Investigation of Metabolic Pathway in Vivo. Healthy
21
22 Sprague-Dawley rats (about 220 g in weight) were purchased from Shanghai SLAC
23
24
25 Laboratory Animal Co.,Ltd, and were divided into four groups and then administered of
26
27
E-A/P-CeO2 dispersed in saline by the tail vein at different doses: control, 5, 10, 20
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30 mg/kg. After a period of 30 days, blood samples for complete blood panel assay and
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33 serum biochemistry test, and tissues (heart, kidney, liver, spleen, lung) for histological
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35 analysis via hematoxylin and eosin (H&E) staining were harvested from rat.
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38 The rats that treated by E-A/P-CeO2 at a dose of 10 mg/kg were fed in metabolism cage,
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respectively. Faeces and urine were collected from the cage at several time points and
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43 then dissolved in aqua regia for one month. The concentrations of Ce in the samples were
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46 measured by ICP-OES, and a small amount of urine was taken to detect whether there
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48 existed E-A/P-CeO2 particles or not by TEM and EDS.
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8 ASSOCIATED CONTENT
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11 Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website
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at http://pubs.acs.org.
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A glossary of abbreviations, additional figures and results as described in the text
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22 Conflict of Interest: The authors declare no competing financial interest.
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25 AUTHOR INFORMATION
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27
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29 Corresponding Author
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31
*E-mail: jlshi@mail.sic.ac.cn (J. L. Shi), huping@mail.sic.ac.cn (P. Hu), lmpan@msn.cn
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34 (L. M. Pan)
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37
38 ACKNOWLEDGMENT
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41 This work was financially supported by National Natural Science Foundation of China
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44 (Grant No. 51502326, 51702349), Shanghai Yangfan Program (16YF1412800), Youth
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46
Innovation Promotion Association Chinese Academy of Sciences (Grant No. 2017299),
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49 Science Foundation for Youth Scholar of State Key Laboratory of High Performance
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52 Ceramics and Superfine Microstructures (SKL201704).
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34 functions of each part on edaravone-carried and PEG/ANG conjugated ceria
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37 nanoparticles (E-A/P-CeO2). (c) Scheme of the synthetic procedure for surface
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39 modification and drug loading. (d) Schematic illustration of receptor-mediated
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42 (ANG-LRP) endocytosis of E-A/P-CeO2, part of which remains inside the BCECs for
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44 BBB-protection (i) and the others cross the BBB via transcytosis for stroke treatment (ii).
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39 modified ceria nanoparticles (A/P-CeO2) dispersed in PBS. (c) EDS spectrum and (d)
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42 XRD pattern of A/P-CeO2. (e) XPS analysis of Ce 3d showing the binding energy (BE)
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44 levels of Ce (III) (885.0 and 903.5 eV) and Ce (IV) (882.1, 888.1, 898.0, 900.9, 906.4,
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47 and 916.4 eV). (f) Release profile of edaravone from E-A/P-CeO2 in phosphate buffer
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48 CLSM images of BCECs incubated with P-CeO2 and E-A/P-CeO2 for 1, 4, 7 h at 50 ppm
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51 of ceria nanoparticles, scale bars: 20 µm. (c) Schematics of the BCECs transwell assay
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56 E-A/P-CeO2 through the BBB model in vitro showing the much more effective
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4 BBB-crossing of E-A/P-CeO2 than that of P-CeO2. Free ANG was used as the blocking
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40 within 60 min in each cycle for two cycles. (c) Viabilities of BCECs incubated with
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43 varied concentrations of E-A/P-CeO2 for 24 h or 48 h. (d) BCECs viabilities by CCK-8
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45 assay after being treated with 50 ppm ceria nanoparticles (P-CeO2, A/P-CeO2,
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48 E-A/P-CeO2) for comparing the protective capabilities of ceria nanoparticles against cell
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50 treatment; (b) Time-dependent concentrations (µg Ce/g brain tissue) of ceria
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52 nanoparticles in normal brain tissue at the injection dose of 0.5 mg/kg of ceria
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55 nanoparticles (*P<0.05). (c) Digital photograph of representative TTC-stained brain in
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42 integrity: (a) Digital photographs of brains stained by permeating EB; (b) Corresponding
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44 representative confocal images (scale bar = 50 µm) of the brain slices showing red
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47 fluorescences of EB under the excitation at wavelength 620 nm. Toxicity studies of
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50 E-A/P-CeO2 in vivo: (c) H&E-stained tissue sections (heart, liver, spleen, lung and kidney)
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52 from rats after intravenous injections of E-A/P-CeO2 at varied doses of 5, 10 and 20
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