Simultaneous BloodâBrain Barrier Crossing and Protection for Stroke

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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 Downloaded from http://pubs.acs.org on June 22, 2018

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Simultaneous Protection

Blood–Brain for

Stroke

Barrier Treatment

Crossing

and

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, 200050, China. ‡

University of Chinese Academy of Sciences, Beijing, 100049, PR China.

§

School of Physical Science and Technology, ShanghaiTech University, Shanghai,

201210, PR China.

* Email addresses: [email protected] (J. L. Shi), [email protected] (P. Hu), [email protected] (L. M. Pan)

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ABSTRACT: Cerebral vasculature and neuronal networks will be largely destroyed due to the oxidative damages by over-produced reactive oxygen species (ROS) during stroke, accompanied by the symptoms of ischemic injury and blood–brain barrier (BBB) disruption. Ceria nanoparticles, acting as an effective and recyclable ROS scavenger, have been showing high effectiveness in neuroprotection. However, the brain access of nanoparticles can only be achieved by targeting the damaged area of BBB, leading to the disrupted BBB unprotected and turbulence of microenvironment in brain. Nevertheless, the integrity of BBB will cause very limited accumulation of therapeutic nanoparticles in brain lesions. This dilemma is a great challenge in the development of efficient stroke nanotherapeutics. Herein, we have developed an effective stroke treatment agent based on monodisperse ceria nanoparticles, which are loaded with edaravone and modified with Angiopep-2 and poly ethylene glycol on their surface (E-A/P-CeO2). The as-designed E-A/P-CeO2 features highly effective BBB-crossing via receptor-mediated transcytosis to access brain tissues and synergistic elimination of ROS by both the loaded edaravone and ceria nanoparticles. Resultantly, the E-A/P-CeO2 with low toxicity and excellent hemo/histocompatibility, can be used to effectively treat strokes by great intracephalic uptake enhancement and in the meantime effective BBB protections, holding great potentials in the stroke therapy with much mitigated harmful side-effects and sequelae. KEYWORDS: stroke, blood–brain barrier, ceria nanoparticles, edaravone, reactive oxygen species


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Stroke is one of the most common causes of disability which ranks No. 5 among all causes of death.1 Currently, the treatments of ischemic stroke mainly include thrombolytic and neuroprotective therapies.2 Due to the highly limited therapeutic time window, only a few patients (2% − 7%) can benefit from effective thrombolytic therapy within 4.5 h post onset.3 Free radical scavengers, as a kind of important neuroprotective agents, can prevent neurons from being damaged in a chain reaction induced by reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide anion (O2-•) and hydroxyl radical (•OH), which are some of the core pathological causes of ischemic stroke.4 Although the effective neuroprotection by edaravone has been proved, several critical problems remain, such as the ineffectiveness in crossing the blood–brain barrier (BBB), repetitive and over-high dosage, and severe side effects. The application of nanobiotechnology, as free radical scavengers,

6-16

provides promising approaches to

achieve efficient treatments of stroke. Ultrasmall ceria nanoparticles can easily lose oxygen and/or electrons in the fluorite lattice structure, which leads to the generation of oxygen vacancies and lowered valence states of cerium atoms, and the resultant antioxidant activity based on electron transfer between cerium (III) and cerium (IV) (Scheme 1a). Besides, due to the memory function of the lattice structure and electron exchange with other ions, ceria nanoparticles will easily recover their redox activity, allowing repetitive elimination of ROS.13-16 As a result, ceria nanoparticles can be applied to mimic antioxidant enzymes (superoxide dismutase17 (SOD) and catalase18 (CAT)) in the treatments of strokes associated with high oxidative


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stresses. However, a number of shortcomings still remain to be overcome: (i) To ensure the high biomimetic enzyme activity,19 ceria nanoparticles of smaller than 5 nm in diameter are more favorable. However, the ultrasmall sizes will lead to over-short vascular circulation half-life, and the easy inter-particle agglomeration of these ultrasmall ceria nanoparticles makes the issue much worse; (ii) Reluctant responses in capturing free radicals of extremely short half-life and free path20,21 due to the over-thick surface-modification of ceria nanoparticles for maintaining biocompatibility and stabilization in physiological environment; (iii) Most importantly, the potential BBB disruption for the sake of effective nanoparticle accumulation in brains. As the exclusive biological barrier, BBB protects the brain from potentially harmful compounds in the blood, but also unfortunately prevents the intracephalic accumulation of nanoparticles.22 Since the BBB could be broken down in brain ischemia,23-25 only a tiny proportion of nanoparticles get access to brain lesion by targeting the damaged BBB area. 26 Thus the therapeutic efficacy for neuroprotection is still greatly limited. And the slow opening of the damaged BBB took several hours, resulting in the extremely low accumulation of nanoparticles in the brain lesion.27,28 More unfortunately, the stroke-induced disruptions of BBB will most likely cause neurological dysfunctions,24 such as acute cerebral edema and hemorrhage. Therefore, developing an elaborate strategy to achieve simultaneous BBB-crossing and protection for stroke treatment, is of great significance. In this study, a kind of BBB-targeting edaravone-loaded ceria nanoparticles has been first developed for simultaneous BBB-crossing and stroke treatment. As illustrated in


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Scheme 1, the designed nanoplatform exhibit a core-shell structure with monodispersed ultrasmall ceria as core, organic Angiopep-2 (ANG)/ poly ethylene glycol (PEG) as shell and edaravone loaded in the organic shell. It consists of three key components: (i) PEG significantly improves the biocompatibility as well as monodispersity, and extends the half-lives in the bloodstream.29 (ii) Edaravone loaded in the shell synergistically scavenges ROS, especially those not close to ceria core. Thus ROS scavenging efficiency was significantly improved. (iii) ANG as the targeting ligand on ceria nanoparticles, which was used to specially bind to the low density lipoprotein receptor-related protein (LRP) overexpressed on cells that comprise the BBB.30,31 On the whole, the BBB-targeting nanoplatform, based on the ceria nanoparticle core/ PEG-ANG shell structure, is able to competently cross BBB via ANG-LRP-mediated transcytosis. During this process, BBB will be protected from oxidative stress-induced disruption in stroke owing to the synergistic elimination of ROS by ceria nanoparticle core and molecular edaravone absorbed in the organic shell. Moreover, in the brain lesion tissues beyond BBB where the nanoplatform has finally reached, ceria cores show highly sustained ROS scavenging capability in addition to the ROS-scavenging effect of edaravone retained in the nanoplatform, thanks to the reproducibility of cerium. Altogether, this work indicates that the nanoplatform based on ANG-conjugated and edaravone-loaded ceria nanoparticles can not only enhance the intracephalic uptake but also prevent the injuries on both brain tissues and BBB in stroke.


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RESULTS AND DISCUSSION Synthesis and Characterization of E-A/P-CeO2. Scheme 1b illustrates the structure of E-A/P-CeO2 nanoparticles, which were synthesized according to the following main steps. First, ceria nanoparticles with oleylamine on the surface (OA-CeO2) were obtained by a catalytic pyrolysis method.32 Subsequently, owing to the strong ligand binding between ceria and carboxyl/thiol groups,30,33 the ceria nanoparticles were decorated with ANG and amine poly (ethyleneglycol)-carboxyl (NH2-PEG1k-COOH) in parallel (A/P-CeO2) through ligand exchange with oleylamine. Finally, the molecular edaravone was loaded in the organic layer on the ceria nanoparticles (E-A/P-CeO2) by van der Waals forces and organic macromolecules absorption. As shown in the transmission electron microscopic (TEM) images (Figure 1a), OA-CeO2 in hexamethylene are uniform and near-spherical with an average diameter of 4.3 ± 0.5 nm. After coated with ANG/PEG, the ceria nanocrystals were transferred from oleic phase into aqueous solutions (Figure 1b), which resulted in excellent dispersibility and quasi-spherical morphology. To verify the successful exchange of ligands, Fourier transform infrared spectroscopy (FT-IR) and Zeta-potential measurements were carried out (Figure S1a, b). The FT-IR spectra show the disappearance of carbon-carbon double bonds of oleylamine along with the appearance of ether linkages of PEG and reinforcement of carbonyl groups of ANG, indicating that PEG and ANG have replaced oleylamine as the coating layer on ceria nanoparticles. These results were further confirmed by the decreased potential by Zeta-potential analysis. And a peak corresponding to sulfur (S) belonging to ANG can be


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observed from the Energy-dispersive X-ray spectrum (TEM/EDS) that further verifies the successful conjugation of the ANG chains (Figure 1c). Moreover, the ceria nanoparticles after surface modification still possess a pure and typical fluorite cubic structure as evidenced by X-ray diffraction (XRD) analysis (Figure 1d). To demonstrate that the final product of small ceria nanoparticles has a higher percentage of cerium (III), the surface amounts of cerium (III) oxide (peaks at 885.0 and 903.5 eV) and cerium (IV) oxide (peaks at 882.1, 888.1, 898.0, 900.9, 906.4, and 916.4 eV) were quantified by X-ray photoelectron spectroscopy (XPS). The results confirm that the slightly yellow ceria nanoparticles have a cerium (III) oxide content of ∼ 34.1% (Figure 1e), which ensures that the ceria nanoparticles possess antioxidant activity so as to mimic superoxide SOD and CAT. The diameters of PEG/ANG modified ceria nanoparticles (A/P-CeO2) based on dynamic light scattering measurements increased to 19.17 ± 5.24 nm (Figure S1b) due to the presence of hydrated layers, which may prevent ROS from reaching the ceria nanoparticle cores directly and then reduce the ROS-scavenging activity because of the very short effective half-life and limited free path of ROS.20,21 To capture those ROS not reaching the core of ceria nanoparticles, edaravone, serving as molecular ROS scavenger, has been absorbed in the organic macromolecule layer (ANG/PEG) by molecular interaction with A/P-CeO2 during stirring for 12 h, and the final product is named as E-A/P-CeO2. After centrifugation, the supernatant and initial edaravone solutions were both diluted ten-folds and measured by UV-Vis absorption spectroscopy (Figure S1c).


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Edaravone loading was quantified by an internal standard method based on the standard curve at the edaravone characteristic peak (λ = 239 nm) (Figure S1d). And the optimized loading amount of edaravone in A/P-CeO2 was 0.083 g/g at the edaravone to A/P-CeO2 concentration ratio of 1.125 : 1. Then, the release profile of the edaravone in E-A/P-CeO2 nanoparticles has been obtained in Figure 1f, which shows very negligible edaravone release (lower than 4% in 24 h) in the physiological condition. The result indicates that most edaravone will not leak out significantly. This means that edaravone molecules loaded in E-A/P-CeO2 will be mostly retained in an intact nanoparticle rather than detached from the E-A/P-CeO2. Enhanced Cellular Uptake and BBB-Crossing in Vitro. To evaluate whether the as-synthesized E-A/P-CeO2 have acquired high enough targeting capability in vitro, the cellular uptake of E-A/P-CeO2 by brain capillary endothelial cells (noted as BCECs), the main component of BBB, were intensively investigated. Due to the overexpress of LRP on cellular membrane, greatly enhanced cellular uptake of the E-A/P-CeO2 in comparison with P-CeO2 was confirmed by both flow cytometry and confocal microscopy. As shown in flow cytometric measurements (Figure 2a), BCECs treated with fluorescent dye Rhodamine B isothiocyanate (RITC) labelled E-A/P-CeO2 show 2.1 ~ 3.6 times higher positive fluorescence signal ratio than those treated with equimolar amounts of RITC labelled P-CeO2 after their incubation for the same time period, especially during first 1 h, indicating much more rapid E-A/P-CeO2 uptake by BCECs than P-CeO2. In addition, confocal microscopic analysis of BCECs by co-culture with these nanoparticles also


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presents the highly consistent results with the flow cytometry (Figure 2b). Strong red fluorescence signal can be observed around the nucleus of the BCECs when treated with E-A/P-CeO2. In contrast, only much weaker fluorescence signal is visible with P-CeO2 under the same conditions. These data clearly confirm the significantly stronger cellular uptake of E-A/P-CeO2 than P-CeO2 by BCECs, both in quantity and rate, which can be attributed to the specific targeting effect of ANG ligands to the LRP receptor on the membrane of BCECs. The E-A/P-CeO2 uptake reached a maximum in 4 h of incubation, ensuring that E-A/P-CeO2 particles can timely protect BCECs against oxidation-induced death in the BBB. According to the literature reports,30,31,34 transwell filters seeded with BCECs will form a compact layer after proliferating, which could then be used to measure the BBB-crossing efficacy of nanoparticles (Figure 2c). The value of Transendothelial Electrical Resistance (TEER) represents the model’s permeability to inorganic ions and is the most sensitive and traditional indicator of BBB function. The obtained TEER values are typically above 200 Ω cm2 during the experiments, which indicate that the BCECs monolayer is in integrity and therefore meets the requirements of a BBB model in vitro30,34. Then we analyzed the Ce contents on the apical side (AP), cellular monolayer (FIL), and basolateral side (BL) by inductively coupled plasma optical emission spectrometry (ICP-OES) after co-incubating in the apical medium with these nanoparticles over 24 h. Figure 2d reveals that 21.0 ± 2.4% of the targeting E-A/P-CeO2 has penetrated across the BBB layer and been detected on the basolateral side. In


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comparison, only 6.2 ± 1.0% of non-targeting P-CeO2 was found under the same condition, indicating the significant targeting effect of E-A/P-CeO2 compared with P-CeO2. To make sure whether the ANG ligands on E-A/P-CeO2 had played a major role in mediating the BBB-crossing transcytosis with LRP receptors or not, typical blocking assay was further employed. High-dose free ANG was added into the apical medium 0.5 h in advance to block the targeting interaction between E-A/P-CeO2 and LRP receptors. Resultantly the E-A/P-CeO2 amount on the basolateral side significantly decreased to 8.9 ± 1.2%, similar to the level of non-targeting P-CeO2 nanoparticles, confirming the positive role of ANG on BBB targeting. These results clearly demonstrate the advantage of E-A/P-CeO2 in transporting across the BBB by endocytosis, resulting in the enhanced intracephalic uptake and guaranteeing the following neuroprotection effect. Regenerable ROS Scavenging and BCECs Protection in Vitro. The electron spin resonance (ESR) spectroscopy (Figure 3a) was used to detect the generated hydroxyl radicals in Fenton reaction by using 5,5'-dimethylpyrroline-1-oxide (DMPO) as the spin trapping agent. The appearance of 1:2:2:1 multiple peaks in the ESR spectrum evidences the presence of DMPO-OH adducts, suggesting the generation of hydroxyl radicals which is in good agreement with the literature report.35,36 Due to the competitive relations between DMPO and ceria nanoparticles in trapping radicals, the decrease of signal intensity will prove the hydroxyl radicals scavenging effect of ceria nanoparticles during the reaction in a mixture containing Fenton agent, ceria nanoparticles and DMPO. Interestingly, the amount of spin adduct sharply decreased upon adding E-A/P-CeO2


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rather than equal molar amounts of A/P-CeO2, confirming that edaravone loaded in A/P-CeO2 could efficiently promote the ROS scavenging. Similarly, the radical scavenging role of ceria nanoparticles was also examined by UV/Vis spectroscopy using methyl violet (MV)37 (Figure S2a). The maximum absorbance of MV (λ = 582 nm) largely decreased because of the carbon-carbon double bond breakage through the electrophilic addition by hydroxyl radical in Fenton reaction. However, the absorption decay of MV was not significant when free radical scavengers A/P-CeO2 (the mixture b) or E-A/P-CeO2 (the mixture a) were added. These results demonstrate that E-A/P-CeO2 can adsorb and eliminate the hydroxyl radicals to protect the MV from oxidation. Meanwhile, the catalytic effect and regenerability of E-A/P-CeO2 during the reaction with H2O2 were evaluated by in-situ Raman spectroscopy excited by 488 nm laser. As shown in Figure 3b, the major peaks centered at 460 cm−1 can be indexed as a symmetric breathing mode of the oxygen atoms around Ce ions related to the ordering in the oxygen sublattice.38 After adding H2O2 into the above system, the major peaks immediately shift to 850 cm−1 and 880 cm−1 while the peak at 460 cm−1 disappears because of the reaction between ceria nanoparticles and H2O2, leading to the generation of O–O stretching of adsorbed peroxide species (O22−) accompanied by defect aggregation and disordering of oxygen sublattice. As the reaction proceeds, the peaks at 850 cm−1 and 880 cm−1 become gradually weaker, while the one at 460 cm−1 intensifies and approximately returns to the original intensity in 1 h. Here, the intensity changes of the 850 cm−1 and 880 cm−1 peaks from their generation back to disappearance are regarded as an anti-oxidation cycle. As


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anticipated, adding H2O2 again, the changes of the peak intensities in the second cycle are almost identical to the first cycle in the Raman spectra, indicating an excellent recyclable antioxidant property of E-A/P-CeO2. To further verify this, E-A/P-CeO2 reacted with H2O2 was also evaluated by transmitting UV-Vis spectroscopy.39 As shown in Figure S2b, after the injection of H2O2 into the aqueous solution of E-A/P-CeO2, transmitting UV-Vis absorption peak shifted to the right and the color gradually became darker, suggesting that cerium (III) on the E-A/P-CeO2 surface has been oxidized to cerium (IV). In the following 10 days, transmitting UV-Vis absorption peak of E-A/P-CeO2 reversible shifted to the left gradually, which implies the regeneration of Ce (III) on the surface of ceria nanoparticles. The results confirm the auto-regenerative and anti-oxidant properties of the E-A/P-CeO2. Furthermore, the amount of cerium (III) oxide on the surface was quantitatively analyzed by XPS. The results in Figure S2d and S2e show that the surface amounts of cerium (III) oxide decrease upon treating with ROS (H2O2 or •OH from Fenton reaction). Moreover, as the ROS concentration increases, the amount of the cerium (III) oxide also decreases. These results further indicate that E-A/P-CeO2 has an excellent capability of capturing and eliminating ROS. Thus, the above behavior indicates that E-A/P-CeO2 can be used as an effective biomimetic antioxidant such as SOD and CAT to eliminate ROS for stroke treatment. The antioxidant activity at cellular level was evaluated on BCECs. First of all, to rule out the materials' cytotoxicity, Cell Counting Kit-8 (CCK-8) assays were carried out (Figure 3c), which demonstrated the negligible cytotoxicity of these E-A/P-CeO2


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nanoparticles against BCECs in vitro. And then, to investigate the protective effect against oxidative stress-induced cell death, BCECs were treated with tert-butyl hydroperoxide (tBHP) that could trigger the generation of large amounts of intracellular ROS,26 and then tested using CCK-8 assay. As shown in Figure 3d, the cell viabilities have been largely reduced after co-incubation with the different doses of tBHP. Once protected by ceria nanoparticles against oxidative stress-mediated injury, the cell survival rate shows no noticeable decrease, but even slight increase at the low concentration (25 ppm, Figure S3). To reveal the protective effects of different kinds of ceria-based nanoparticles, cells incubated with P-CeO2, A/P-CeO2, E-A/P-CeO2 nanoparticles were tested. The higher cell viability with A/P-CeO2 than that with P-CeO2 demonstrates the contribution of ANG-targeting in enhancing the cellular uptake, which efficiently and timely eliminates intracellular ROS. The highest cell viability by adding E-A/P-CeO2 further indicates that edaravone loaded in A/P-CeO2 could synergistically elevate the ROS scavenging activity. For further quantitative analysis, Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) Apoptosis Detection kit was used to measure the BCECs apoptosis ratio by flow cytometer (Figure 3e). It can be found that E-A/P-CeO2 shows the highest percentage of live cells (41.1%) and the lowest percentage of late apoptosis cells (25.0%) among the samples, in comparison to those of A/P-CeO2 (37.9% live and 31.5% late apoptosis) and P-CeO2 (19.7% live and 34.6% late apoptosis). All the above results demonstrate that E-A/P-CeO2 is capable of effectively protecting BCECs and BBB


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against ROS attack by efficiently eliminating intracellular ROS owing to the enhanced cellular uptake, and the synergistic ROS-scavenging functions by both loaded edaravone in the outer shell and the ceria core in the constructed nanoplatform, which could guarantee its further in vivo application in mitigating oxidative stress and preventing cerebral lesion. BBB-Crossing Transport and Protection against Stroke in Vivo. In order to demonstrate the BBB-crossing capability, cerebral uptake experiments of the ceria nanoparticles were carried out on healthy rats, whose BBB was complete to exclude the effect of damaged BBB in stroke. After intravenous administration, amounts of ceria nanoparticles in brain tissue were tested by ICP-OES (Figure 4b). Compared with P-CeO2, E-A/P-CeO2 shows multifold-enhanced accumulation in brain region in 24 h after injection, indicating that E-A/P-CeO2 nanoparticles have been effectively delivered across the BBB into the brain by an ANG targeting and LRP receptor mediated transcytosis process in vivo. Furthermore the excellent monodispersity and ultrasmall size also contribute to the cerebral accumulation of the nanoparticles. Meanwhile, the bio-distribution of E-A/P-CeO2 in main tissues (heart, lung, kidney, spleen and liver) were also observed (Figure S7a). Although a large majority of E-A/P-CeO2 has accumulated in spleen and liver, the quantity of ceria nanoparticles in per gram brain tissue is still much higher than the others. Such a largely enhanced intracerebral uptake capability of the targeting E-A/P-CeO2 delivery system may offer promising opportunities for in vivo cerebral disease therapy.


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Furthermore, to explore the therapeutic effect of ceria nanoparticles in vivo, the focal cerebral ischemic injury of rat was tested using middle cerebral artery occlusion (MCAO) mode40 by tail vein injection. After stroked for 24 h, the cerebral infarct volume was measured by triphenyltetrazolium chloride (TTC) staining, showing the stained normal tissue in red and the unstained infarction in white. The result shows that the infarct volume of the control group of saline injection is as high as 45.6 ± 4.8%. However, when treated with varied doses of E-A/P-CeO2, the infarct volumes considerably decrease down to 15.0 ± 4.1% at an optimal concentration of 0.6 mg/kg (Figure S4a,b). We further compared the neuroprotective effects of P-CeO2, A/P-CeO2, and E-A/P-CeO2 at the same injection dose of 0.6 mg/kg (Figure 4c). Owing to the enhanced accumulation in brain tissue via ANG targeting, A/P-CeO2 shows more significant neuroprotective effect than P-CeO2, and comparatively E-A/P-CeO2 performs the best, which can be ascribed to the synergistic ROS-scavenging effects by the edaravone component loaded in the targeting systems and the ceria core for the largely improved treatment effect in vivo. Meanwhile, considering that the oxidative damage induced by ROS was the major cause of stroke injury, fluorescent probe of 2,7-Dichlorodi–hydro fluorescein diacetate (DCFH-DA) was used to detect ROS levels in the brain after ischemia induction for the further investigation of antioxidant protective effect of E-A/P-CeO2. ROS in frozen brain sections could oxidize non-fluorescent DCFH to produce red fluorescent DCF based on confocal configuration detection. As shown in the image (Figure 4d), the ROS level in the E-A/P-CeO2 nanoparticles injected brain is markedly reduced compared to the stroke


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control, indicating the highly efficient ROS elimination by E-A/P-CeO2. All the above in vivo results demonstrate that E-A/P-CeO2 are capable of efficient accumulation in brain tissue by BBB-crossing instead of BBB damaging, which is expected to resultantly enhance the therapeutic efficacy of stroke by mitigating the intracerebral oxidative stress. Prevention of the BBB Damage in Vivo. According to the previous reports,24,25 ischemia-hypoxia in stroke would result in cell apoptosis, increased endothelial cell permeability and disassembly of tight proteins and intercellular junctions, which consequently lead to BBB disruption. As a natural defense system to maintain the stability of the brain micro-environment, the damage of BBB is reversible during early stage but will become permanent inflammatory several days later, which might cause the more severe lesions such as vasogenic cerebral edema and hemorrhagic transformation. Thus, it is highly important to protect the BBB integrity in the ischemic stroke treatment. It has been found in this study that the as-synthesized E-A/P-CeO2 features high BBB-crossing capability via receptor-mediated (ANG-LRP) endocytosis, during which part of the E-A/P-CeO2 nanoparticles may remain in BCECs to eliminate the oxidative stress in BBB induced by ischemia-hypoxia as discussed above, and thus BBB integrity can be protected. To verify this, the traditional Evans Blue (EB) staining assay,41 in which EB cannot penetrate through the complete BBB to stain brain tissue, was performed to test the permeability of BBB by measuring the amount of extravasated EB. As shown in the digital photographs (Figure 5a), compared with normal rat (sham), the right half of the brain in control group presents bright blue color after ischemic injury, demonstrating


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that EB has clearly extravasated across the BBB into brain tissue. All ceria nanoparticles-treated groups (P-CeO2, A/P-CeO2, E-A/P-CeO2) show much less significant EB staining in the right halves than the control of stroke. More importantly, the brain EB staining of E-A/P-CeO2-treated rat is the slightest. In accordance with the results of EB fluorescent signal detection in brain slices by confocal microscopy (Figure 5b), the brain tissue of E-A/P-CeO2-treated rat shows the weakest red fluorescence of EB. Quantitative analysis (Figure S5a) of EB exudation from blood into the brain by spectrophotometer (Figure S5b) shows that the amount of EB uptake in brain of E-A/P-CeO2-treated rats is the lowest. The above data clearly indicate that the E-A/P-CeO2-treated rats have maintained the highest level of BBB integrity among all groups tested, demonstrating that E-A/P-CeO2 featuring the ANG-targeting effect can effectively accumulate within the cerebral microvascular endothelial cells and eliminate ROS therein to prevent BBB from breakdown under the attack by oxidase-generated superabundant ROS.42, 43 The long-Term Toxicity of E-A/P-CeO2 in Vivo. Although ceria nanoparticles, possessing a wide range of potential biomedical applications, have attracted extensive interests, however, its potential toxicity in treatment of stroke is still unclear to date16. Herein, toxicity studies using Sprague-Dawley rats were performed. The rats after intravenous injections of E-A/P-CeO2 nanoparticles in different dosage groups (0, 5, 10, 20 mg/kg) for 30 days were euthanized. The blood was acquired by abdominal aortic method for biochemistry and hematology tests, while hematoxylin and eosin (H&E)


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stained main organs for histological analysis. The H&E staining results of major organs (heart, liver, spleen, lung, and kidney) show no significant acute, chronic pathological toxicity, and adverse effects among the control group and the treatment groups (Figure 5c). A series of blood indexes including key biochemistry parameters and organs function, in the drug treatment groups show no abnormities compared to those of the control group (Figure S6). These results preliminarily prove that the E-A/P-CeO2 exhibits no significant appreciable toxicity in vivo. Besides, effective clearance of these nanoparticles from the body is another key factor for biosafety requirements in vivo. To investigate the metabolic pathway of E-A/P-CeO2, feces and urine of the treated rats were collected for an entire week and quantitatively detected by ICP-OES. As shown in Figure S7a-b, only a small fraction of excretion was through kidney into urine, which was further confirmed by TEM and corresponding EDS (Figure S7c) examinations of a random urine sample. The vast majority of E-A/P-CeO2 was excreted via feces due to the larger hydrodynamic diameter than the renal filtration threshold of 5.5 nm. About 7.0% and 49.6% of the E-A/P-CeO2 nanoparticles were cleared by urine and feces within a week, respectively, which exhibit that these nanoparticles could be well excreted from the body to ensure the high biosafety.

CONCLUSIONS In summary, a promising neuroprotective nanoplatform, E-A/P-CeO2 composed of monodispersed ultrasmall ceria cores, organic PEG and ANG shell and edaravone loaded


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in the shell, has been successfully constructed and evaluated. It has been demonstrated that such a nanoplatform is capable of effective BBB crossing thanks to the specific interaction between ANG peptide and LRP receptor. Consequently, BBB will be protected against the ROS attack due to synergistic ROS scavenging by both loaded edaravone in the outer shell and the ceria core. In the meantime, the timely treatment of ROS-induced brain injury in stroke will be achieved by extravasated nanoplatforms. On one hand, E-A/P-CeO2 is a targeting nanodrug capable of active BBB crossing via receptor-mediated transcytosis instead of passive diffusion through the damaged area of BBB for stroke treatment. On the other hand, it is also an effective antioxidant agent for preventing BBB against breakdown in stroke, providing a promising strategy to overcome the contradiction between maintaining BBB integrity to ensure the brain micro-environment stability and the efficient nanoparticle extravasation across BBB for highly accumulation in intracerebral lesions. Meanwhile, low toxicity and good hemo/histocompatibility of E-A/P-CeO2 have also been demonstrated. Therefore, this work highlights an efficient strategy for the targeted treatment for brain nerve lesions and the concurrent BBB protection against damages caused by stroke as well, and may shed a light on other neurodegenerative disease treatments.

METHODS Materials. Cerium (III) nitrate hexahydrate (Ce(NO3)3·6H2O, 99%), oleylamin (OA, 70%),

1-octadecene

(ODE,

90%),

cyclohexane


(99.5%),

and

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5,5-dimethyl-1-pyrroline-N-oxide (DMPO, 98%) were purchased from Sigma-Aldrich. NH2-PEG1k-COOH

were

acquired

from

Jenkem

Co,

Ltd.

Angiopep-2

(TFFYGGSRGKRNNFKTEEYC) was synthesized by Chinese Peptide Company. Edaravone was purchased from J&K scientific. Methyl violet (MV), hydrogen peroxide (H2O2, 30%), Ferrous sulfate heptahydrate (FeSO4·7H2O, 99%) were obtained from Sinopharm Chemical Reagent Co, Ltd. Synthesis of Ceria Nanoparticles.32 First, ceria nanoparticles from CeO2-Oleylamine were prepared through the reported method. Ce(NO3)3•6H2O as the biocompatible precursor44,45 (0.43 g, 1.0 mmol) and oleylamine (0.802 g, 3.0 mmol) were added into 1-octadecene (ODE 4 g). The mixed solution was gently stirred for 1 h at room temperature, and then put at 80 °C for 2 h in an argon atmosphere. Following heated and maintained at 260 °C for 1.5 h. After cooled, the yellow precipitated ceria nanoparticles dissolved in 5 ml hexamethylene and added ethanol or acetone using centrifugation (20,000 rpm for 20 min) for three times. The resulting nanoparticles were dispersible in 20 ml hexamethylene. Subsequently, the ceria were decorated with Angiopep-2 (TFFYGGSRGKRNNFKTEEYC)

and

aminepoly-(ethylene

glycol)-carboxyl

(NH2-PEG1k-COOH) through ultrasound. ANG (4 mg) and PEG (15 mg) were dissolved in 10 mL of ultrapure water. Next 1 mL of ceria nanoparticles/ethyl ether solution (3 mg/mL) was added, which resulted in the ANG/PEG substitution for oleylamine ligand, i.e., hydrophobic ceria being transfered into aqueous phase, under the ultrasonic treatment. A clear faint yellow solution (A/P-CeO2/water) was obtained by stirring at


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room temperature for 24 h to evaporate the methanol. The ceria nanoparticles were collected by ultra-centrifugation and washed with deionized water several times to remove excess ANG and PEG. And then, purified A/P-CeO2 nanoparticles were dispersed in deionized water and calculated the molality at 1.6 mM by ICP-OES. Edaravone (5 mL) with different concentration (0.6, 1.2, 1.8, 2.4 mM) was respectively added to 5 mL of A/P-CeO2/water, and the resulting solution was stirred for 12 h. The mixture and the supernatant by ultra-centrifuged (20,000 rpm for 120 min) were collected and tested by UV-vis, respectively. The loading efficiency of edaravone from E-A/P-CeO2 were calculated as follows: (Wini-Wsup)×100%/WCe (Wini: initial amount of edaravone and Wsup: supernatent amount of edaravone). For assessing the drug release behavior, E-A/P-CeO2 nanoparticles were encapsulated in a dialysis bag of cutoff molecular weight at 3500, and placed into 30 ml releasing medium (pH = 7.4), which were performed on the 37 °C shaking table at shaking speed of 100 rmp. At certain time intervals, the 3 mL releasing medium was taken out and tested by UV-vis at 239 nm to get the released edaravone concentration, then poured back into the medium. The drug-releasing percentage is calculated as follows: Wrel × 100%/Wini (Wrel: the released amount of edaravone, Wini: the initial amount of edaravone). Characterization. Transmission electron microscope (TEM) and energy dispersive X-ray spectroscopy (EDS) on JEM-2100F electron microscope were used to detect morphology and elemental distribution analysis with a voltage of 200 kV. The X-ray


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diffraction (XRD) was recorded by a Rigaku D/MAX-2250 V diffractometer with a Cu Kα radiation target (40 kV, 40 mA). X-ray photoelection spectroscopy (XPS) on a VGMicro MK II instrument was operated with monochromatic Mg Ka X-rays (150 W, 1253.6 eV). FTIR spectra on an IRPRESTIGE-21 spec-trometer (Shimadzu) were measured in the attenuated total reflectance methods. UV–vis absorption and transmission spectra were recorded on UV-3101PC Shimadzu spectroscope. Zeta potential and hydrodynamic size were tested by Dynamic light scattering (DLS Malvern ZetasizerNanoseries ZS90). Inductively coupled plasma optical emission spectrometry (ICP-OES, Agilent 725, Agilent Technologies, US) was used to analyze Ce concentrations of samples. The confocal microscopy (CLSM) images were recorded on FV1000, Olympus, Japan. The flow cytometry (BD LSRFortessa) was utilized to analyze cellular uptake and apoptosis. Enhanced Cellular Uptake in Vitro. 5 mg ceria nanoparticles (P-CeO2, A/P-CeO2, E-A/P-CeO2) were respectively mixed with 0.5 mg RITC in 5 ml water and the solutions were stirred for 12 h. After ultracentrifugation, RITC-conjugated ceria nanoparticles were dispersed in DMEM at a concentration of 50 ppm. BCECs were seeded into 6-well microplates with a density of 105/well and allowed to adhere overnight at 37 °C under 5% CO2. And then the culture medium was changed with as-prepared DMEM containing ceria nanoparticles. After co-incubation for 1, 4 or 7 h, the BCECs were rinsed with PBS three times to remove the non-uptaken ceria nanoparticles, and tested by a flow cytometry analysis in RPhycoerythrin. Meanwhile, BCECs were seeded into a


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CLSM-special cell culture dish and treated by the process described above, and then stained with DAPI. Cellular uptake of ceria nanoparticles was detected by the confocal fluorescence on an Olympus FV1000 laser-scanning microscope under the excitation at 358 nm for DAPI and 540 nm for RITC. BBB-Crossing Model in Vitro. The BBB model was constructed by polycarbonate 24-well Transwell membrane of 1.0 mm mean pore size, 0.33 cm2 surface areas (FALCON Cell Culture Insert, Becton Dickinson Labware, USA), in which BCECs were seeded at a density of 104 cells/well and cultured for 4 days. Epithelial voltohmmeter (Millicell-RES, Millipore, USA) was used to measure the transendothelial electrical resistance (TEER) of cell monolayers, and those of above 200 Ω cm2 were selected for experiments. The ceria nanoparticles (P-CeO2, E-A/P-CeO2 particles) diluted in Roswell Park Memorial Institute medium (RPMI) 1640 at the concentration of 50 ppm were added into the apical side chamber (imitating blood side in vivo) with 50 rpm shaking at 37 °C for 18 h. Finally, the apical side chamber, the filter membrane and the basolateral medium were collected and analyzed of the Ce contents by ICP-OES. To further evidence the transcytosis mechanism by ANG-mediated, a blocking study was carried out in parallel by adding free ANG at a concentration of 3 mg/mL to the apical side chamber 0.5 h before adding E-A/P-CeO2 particles, followed by the identical above-mentioned steps. Evaluation of the Antioxidation and Reproducibility in Vitro. ROS detection by electron spin resonance (ESR) spectroscopy: 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was selected as the spin trapping agent (5 µL, 98%) in ESR spectrum (Bruker EMX1598


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spectrometer) for evaluating the effect of ceria nanoparticles (100 µL, 200 ppm) in scavenging free radical that produced in Fenton reaction (FeSO4: 70 µL, 0.735mM and H2O2: 25 µL, 0.315 mM) in the sample of 200 µL of the total volume. The effects of hydroxyl radicals elimination were related with the change of relative peak intensity of the ESR spectra of DMPO-OH·adducts. ROS detection by UV-vis spectroscopy: hydroxyl radical scavenging capability of ceria nanoparticles was further detected by a photometric method in a system containing methyl violet (MV) and Fenton agent. The sample containing 0.012 mM MV, 0.15 mM FeSO4, 1.0 M H2O2, and an appropriate amount of ceria nanoparticles in phosphate buffer solution (PBS, pH = 7.4) of 5 mL of total volume. After incubation in dark for 5 min, the ultraviolet absorption of the reaction solutions was measured, and the antioxidation performance was evaluated by comparing the maximum absorbance of MV in the conditions with and without ceria nanoparticles. Detection by Raman spectra: the sample solution containing 10 mg ceria nanoparticles and 10 mg H2O2 in 0.1 mL distilled water was dropped on the glass sheet and measured by Laser Raman Microscope (Thermo) under 488 nm laser excitation. The Raman spectra of the as-prepared sample were obtained in the time course (0, 10, 30, 60 min). Immediately after the first cycle, 10 mg H2O2 in 0.1 mL distilled water was dropped on the previous sample again and the examination process was the same as the above. Cytotoxicity Assessment in Vitro. Brain capillary endothelial cells (noted as BCECs, Cellbio (Shanghai) Ltd) were maintained at 37 °C and with 5% CO2 in Dulbecco’s


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Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin in a humidified incubator. Cells were generally plated in cell culture flask (Corning, USA) and allowed to adhere for 24 h then harvested by treatment with 0.25% trypsin-EDTA solution (Gibco, USA). BCECs viability was evaluated by a standard CCK-8 assay (Cell Counting Kit, Beyotime Institute of Biotechnology, Shanghai, China), which were seeded into 96-well tissue culture plates at the density of 106/well and then incubated for 24 h at 37 °C and 5% CO2 to ensure the cell attach. Afterwards, The culture medium was changed with fresh medium containing ceria nanoparticles at different Ce concentrations (0, 3.125, 6.25, 12.5, 25, 50, 100, 200 ppm), In 24 h or 48 h, the original medium was removed, CCK-8 solutions were added to each well of the microtiter plate, incubated for another 1 h in a CO2 incubator, the absorbance of formazan in each well was measured by an enzyme-linked immunosorbent assay reader (BioTeck instrument, USA) at 490 nm to calculate the cell viability to the control group. Protection against Intracellular ROS in Vitro. BCECs were grown in 96-well culture medium plates at the density of 106/well for 24 h at 37 °C and 5% CO2, and then the solution of tert-butyl hydroperoxide (tBHP) as an oxidant was added into each well. In 1.5 h, BCECs were washed with PBS three times and new media containing P-CeO2, A/P-CeO2, or E-A/P-CeO2 (25 or 50 ppm) were added into to each well. In 6 h after the treatments, standard CCK-8 assay was performed for viability tests. Meanwhile, BCECs were also seeded into 6-well microplates at a density of 105/well and treated by the steps described above. Finally, after co-incubation with ceria nanoparticles for 6 h, the BCECs


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were immediately resuspended in 500 µl of binding buffer and stained by 2 µM Annexin V-FITC for 15min, 4.5 µM propidium iodide (PI) for 10 min in the dark (Annexin V-FITC/PI Apoptosis Detection Kit, Beyotime). the cell apoptosis was examined using flow cytometry (BD LSRFortessa). Transport across BBB and Distribution of Ceria Nanoparticles in Vivo. Healthy Sprague-Dawley rats (about 220 g in weight) were purchased from Shanghai SLAC Laboratory Animal Co.,Ltd. And all animal studies were based on the guidelines by the Institutional Animal Care and Use Committee. P-CeO2 and E-A/P-CeO2 were respectively dispersed in saline solution after ultraviolet disinfection, and then were injected into the two groups of rats by the tail vein with a dose of 0.5 mg/kg. After 24 h, the brains, hearts, kidneys, livers, spleens, lungs were rapidly acquired and weighed up after decapitating the rats. The samples of tissues dissolving in aqua regia for one month were obtained to measure the concentrations of Ce by using ICP-OES.

Protection against Stroke in Vivo. According to the previous reports, ischemic stroke was successfully induced by constructing Middle Cerebral Artery Occlusion (MCAO) Model. Subsequently, E-A/P-CeO2 particles dispersed in saline solution were intravenously injected into rats at varied doses of 0, 0.3, 0.6, 0.9 mg/kg. In 24 h, these rats were perfused with saline, then, their brains were quickly harvested and stained by 2,3,5-triphenyltetrazolium chloride (TTC). Finally, these brains were cut into five slices


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after immediate freezing, and photos were taken using digital camera. The normal tissues were stained red while the ischemia tissues did not produce change as shown pale. The percentage of infarct volumes were analyzed by Image-Pro Plus. To compare the effects among P-CeO2, A/P-CeO2, E-A/P-CeO2, these nanoparticles were respectively intravenously injected into different rats by the above process at the same dose. To further testify the effect of ceria nanoparticles on inhibiting cell apoptosis in vivo, Reactive Oxygen Species Assay Kit (ROS assay kit based on DCFH-DA) was used to mark the brain area with DAPI being used as a counter stain in nucleus, and then kept at 37 °C for 30 min in dark place. By Fluorescent Microscopy test, DAPI-stained nuclei became blue (emission monitored at 420 nm) under UV excitation at 330-380 nm, and ROS positive cells labeled by fluorescein were red (emission monitored at 590 nm) under excitation at 510-560 nm. Prevention of the BBB Damage in Vivo. The rats were divided into five groups. One group was healthy as sham-operated group, others were quickly treated with saline, P-CeO2, A/P-CeO2 and E-A/P-CeO2 particles at the same dose in 22 h of stroke. Then each group was subject to the following steps of experiments. Briefly, in 22 h of stroke, two percent EB was dissolved in saline and injected into rats as a BBB permeability tracer at the dose of 4 ml/kg. After circulation for 2 h, the rat chest walls were opened to perfuse with saline through the left ventricle. The brains were carefully removed and weighed up, then, were taken photos using a digital camera. Meanwhile, the brain tissue was frozen and sliced of around 10 µm in thickness, and the permeability of EB was


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observed at 680 nm under a confocal laser scanning microscope under the excitation at 620 nm. To further quantify the amount of EB, the brain was cut into small fragments and incubated in dimethyl formamide for 24 h at 60 °C. The homogenate of the brain was centrifuged and the supernatant was taken out for absorbance measurement at 620 nm by the microplate reader, then, the content of the extraction of EB was calculated via standard concentration line. Toxicity Assay and Investigation of Metabolic Pathway in Vivo. Healthy Sprague-Dawley rats (about 220 g in weight) were purchased from Shanghai SLAC Laboratory Animal Co.,Ltd, and were divided into four groups and then administered of E-A/P-CeO2 dispersed in saline by the tail vein at different doses: control, 5, 10, 20 mg/kg. After a period of 30 days, blood samples for complete blood panel assay and serum biochemistry test, and tissues (heart, kidney, liver, spleen, lung) for histological analysis via hematoxylin and eosin (H&E) staining were harvested from rat. The rats that treated by E-A/P-CeO2 at a dose of 10 mg/kg were fed in metabolism cage, respectively. Faeces and urine were collected from the cage at several time points and then dissolved in aqua regia for one month. The concentrations of Ce in the samples were measured by ICP-OES, and a small amount of urine was taken to detect whether there existed E-A/P-CeO2 particles or not by TEM and EDS.


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ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. A glossary of abbreviations, additional figures and results as described in the text Conflict of Interest: The authors declare no competing financial interest. AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected] (J. L. Shi), [email protected] (P. Hu), [email protected] (L. M. Pan)

ACKNOWLEDGMENT

This work was financially supported by National Natural Science Foundation of China (Grant No. 51502326, 51702349), Shanghai Yangfan Program (16YF1412800), Youth Innovation Promotion Association Chinese Academy of Sciences (Grant No. 2017299), Science Foundation for Youth Scholar of State Key Laboratory of High Performance Ceramics and Superfine Microstructures (SKL201704).


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Disorders. Neuron 2008, 57, 178-201. 28. Saraiva, C.; Praça, C.; Ferreira, R.; Santos, T.; Ferreira, L.; Bernardino, L. Nanoparticle-Mediated Brain Drug Delivery: Overcoming Blood–Brain Barrier to Treat Neurodegenerative Diseases. J. Control. Release 2016, 235, 34-47. 29. Rivera-Gil, P.; Aberasturi, D. J. D.; Wulf, V.; Pelaz, B.; del Pino, P.; Zhao, Y.; de Ia Fuente, J. M.; Ruiz de Larramendi, I.; Liang, X. J.; Parak, W. J. The Challenge to Relate the Physicochemical Properties of Colloidal Nanoparticles to Their Cytotoxicity. Acc. Chem. Res. 2012, 46, 743-749. 30. Ni, D.; Zhang, J.; Bu, W.; Xing, H.; Han, F.; Xiao, Q.; Yao, Z.; Chen, F.; He, Q.; Liu, J.; Zhang, S.; Fan, W.; Zhou, L.; Peng, W.; Shi, J. Dual-Targeting Upconversion Nanoprobes across the Blood-Brain Barrier for Magnetic Resonance/Fluorescence Imaging of Intracranial Glioblastoma. ACS Nano 2014, 8, 1231-1242. 31. Sun, X. Y.; Pang, Z. Q.; Ye, H. X.; Qiu, B.; Guo, L. R.; Li, J. W.; Ren, J. F.; Qian, Y.; Zhang, Q. Z.; Chen, J.; Jiang, X. G. Co-Delivery of pEGFP-hTRAIL and Paclitaxel to Brain Glioma Mediated by an Angiopep-Conjugated Liposome. Biomaterials 2012, 33, 916–924. 32. Lee, S. S.; Zhu, H.; Contreras, E. Q.; Prakash, A.; Puppala, H. L.; Colvin, V. L. High Temperature Decomposition of Cerium Precursors to Form Ceria Nanocrystal Libraries for Biological Applications. Chem. Mater. 2012, 24, 424-432. 33. Zgheib, N.; Putaux, J. L.; Thill, A.; Bourgeat-Lami, E.; D'Agosto, F.; Lansalot, M. Cerium Oxide Encapsulation by Emulsion Polymerization Using Hydrophilic


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Scheme 1. (a) Schematics showing the formations of oxygen vacancies, cerium (III) and cerium (IV) species in ceria nanoparticles as biomimic antioxidant enzymes. (b) Main functions of each part on edaravone-carried and PEG/ANG conjugated ceria nanoparticles (E-A/P-CeO2). (c) Scheme of the synthetic procedure for surface modification and drug loading. (d) Schematic illustration of receptor-mediated (ANG-LRP) endocytosis of E-A/P-CeO2, part of which remains inside the BCECs for BBB-protection (i) and the others cross the BBB via transcytosis for stroke treatment (ii).


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Figure 1. TEM (scale bar = 50 nm) and HTEM (scale bar = 5 nm) images of (a) oleylamine-coated ceria nanoparticles dispersed in cyclohexane, and (b) PEG/ANG modified ceria nanoparticles (A/P-CeO2) dispersed in PBS. (c) EDS spectrum and (d) XRD pattern of A/P-CeO2. (e) XPS analysis of Ce 3d showing the binding energy (BE) levels of Ce (III) (885.0 and 903.5 eV) and Ce (IV) (882.1, 888.1, 898.0, 900.9, 906.4, and 916.4 eV). (f) Release profile of edaravone from E-A/P-CeO2 in phosphate buffer saline (pH 7.4) during a period of 24 h. And from 16 h to 24 h, the release percentages remained almost constant at 3.32 ± 0.10%.


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Figure 2. (a) Flow cytometry analysis of BCECs as the negative control, and BCECs after co-incubated with RITC-labeled P-CeO2 and E-A/P-CeO2 for 1, 4, 7 h in vitro. (b) CLSM images of BCECs incubated with P-CeO2 and E-A/P-CeO2 for 1, 4, 7 h at 50 ppm of ceria nanoparticles, scale bars: 20 µm. (c) Schematics of the BCECs transwell assay for constructing BBB model in vitro. (d) Relative transcytosis amounts of P-CeO2 and E-A/P-CeO2 through the BBB model in vitro showing the much more effective


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BBB-crossing of E-A/P-CeO2 than that of P-CeO2. Free ANG was used as the blocking agent, displaying the significant role of ANG-targeting in BBB-crossing of E-A/P-CeO2 nanoparticles. *P

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