Bioengineered Boronic Ester Modified Dextran Polymer Nanoparticles

Jun 5, 2018 - ... neurons after homing to ischemic brain tissues. The potential of the SHp-RBC-NP for ischemic stroke therapy was systematically evalu...
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Bioengineered Boronic Ester Modified Dextran Polymer Nanoparticles as Reactive Oxygen Species Responsive Nanocarrier for Ischemic Stroke Treatment Wei Lv,†,§,# Jianpei Xu,†,# Xiaoqi Wang,† Xinrui Li,‡ Qunwei Xu,† and Hongliang Xin*,† †

Department of Pharmaceutics, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China Sir Run Run Hospital, Nanjing Medical University, Nanjing 211166, China § Jiangsu Jiangyin People’s Hospital, Jiangyin 214400, China ‡

S Supporting Information *

ABSTRACT: Ischemic stroke is a leading cause of long-term disability and death worldwide. Current drug delivery vehicles for the treatment of ischemic stroke are less than satisfactory, in large part due to their short circulation lives, lack of specific targeting to the ischemic site, and poor controllability of drug release. In light of the upregulation of reactive oxygen species (ROS) in the ischemic neuron, we herein developed a bioengineered ROS-responsive nanocarrier for strokespecific delivery of a neuroprotective agent, NR2B9C, against ischemic brain damage. The nanocarrier is composed of a dextran polymer core modified with ROS-responsive boronic ester and a red blood cell (RBC) membrane shell with stroke homing peptide (SHp) inserted. These targeted “core−shell” nanoparticles (designated as SHp-RBC-NP) could thus have controlled release of NR2B9C triggered by high intracellular ROS in ischemic neurons after homing to ischemic brain tissues. The potential of the SHp-RBC-NP for ischemic stroke therapy was systematically evaluated in vitro and in rat models of middle cerebral artery occlusion (MCAO). In vitro results showed that the SHp-RBC-NP had great protective effects on glutamate-induced cytotoxicity in PC-12 cells. In vivo pharmacokinetic (PK) and pharmacodynamic (PD) testing further demonstrated that the bioengineered nanoparticles can drastically prolong the systemic circulation of NR2B9C, enhance the active targeting of the ischemic area in the MCAO rats, and reduce ischemic brain damage. KEYWORDS: (Ischemic stroke, Biomimetic, Stimuli-responsive nanocarrier, Stroke homing peptide, Neuroprotectant)

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could thus deliver therapeutics in a spatially and temporally controlled manner. Among these ROS-labile groups, boronic ester conjugated with copolymer was reported as an intelligent ROS-responsive biomaterial to fabricate nanoparticles by selfassembly.3,4 Therefore, we hypothesized that there could be a great potential to use boronic ester modified polymers to develop ROS-responsive nanocarriers for the treatment of ischemic stroke. Red blood cell (RBC)-biomimetic engineered delivery systems have massively emerged in drug delivery field recently.5−9 For example, tissue-type plasminogen activator has been coupled to the RBC to develop a pharmaceutical approach for the treatment of ischemic stroke.10−12 Impor-

schemic stroke is recognized as one of the most serious public health problems. Thus, there is an urgent need to design and develop smart drug carriers with desirable physicochemical and biological properties such as long circulation time, favorable specific targeting capability to the ischemic site, and good controllability of drug release for the enhanced treatment of ischemic stroke. It is well-known that after the reperfusion, enormous toxic reactive oxygen species (ROS) are upregulated in the ischemic site, leading to the injury of neurons.1 However, from the pharmaceutical drug delivery design perspective, the elevated levels of ROS in the ischemic site have the potential to be used as a smart sensitive trigger for controlled drug release, which could be applied for the development of site-specific drug delivery systems. Recently, some advanced nanomaterials have been designed for stimuli response to intracellular oxidative conditions by incorporation of ROS-labile groups, such as boronic ester, proline, and thioketal.2 These ROS-responsive nanocarriers © XXXX American Chemical Society

Received: January 18, 2018 Accepted: May 31, 2018

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DOI: 10.1021/acsnano.8b00477 ACS Nano XXXX, XXX, XXX−XXX

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Figure 1. Schematic design of the SHp-RBC-NP/NR2B9C. After intravenous injection, the SHp-RBC-NP/NR2B9C could prolong the circulation life with the RBC-mimicking properties and then target to the ischemic brain site via stroke homing peptide mediated transcytosis. When internalized into ischemic neurons, the NR2B9C is released from the PHB-dextran polymer nanoparticles attributable to the high levels of intracellular ROS and then selectively disrupted the NMDARs with PSD-95 to prevent the overproduction of nitric oxide (toxic signaling agent).

to prevent the overproduction of nitric oxide (toxic signaling agent) without blocking the normal NMDAR functions (synaptic activity or calcium influx).15,16 With the RBCmimicking properties, these nanoparticles exhibited a prolonged circulation life. The stroke homing peptide also enhanced the active targeting capability of the nanoparticles to the ischemic site. When the bioengineered, targeted, ROSresponsive nanoparticles were actively delivered to the ischemic area, the NR2B9C was released in neurons from the degraded nanoparticles attributable to the high levels of intracellular ROS, thus achieving effective therapeutic efficacy for ischemic brain.

tantly, the RBC membrane also plays a significant role in camouflaging exogenous nanoparticles and enables nanoparticles with advanced natural characteristics including superbiocompatibility, better immune-evading capabilities, and prolonged blood retention as compared to their PEGylated counterparts.5,6 Furthermore, the hydrophilic glycans and the negatively charged sialic acid residues on the surface of RBC membranes would contribute to the structural formation of the mimetic RBC membrane camouflaged nanoparticles (RBCNPs) as well as the long blood circulation characteristics.7 For active drug delivery in the treatment of ischemic stroke, a stroke-homing peptide (SHp, CLEVSRKNC) was identified and optimized by in vivo phage display in a focal cerebral ischemia rat model.13 It was found that the CLEVSRKNC peptide could selectively target to the ischemic site in brain and colocalize to a portion of neuronal cells undergoing apoptosis at the penumbra region of ischemic brain tissue. Moreover, SHp modified liposome was reported to be able to effectively home to the ischemic stroke site.14 In this study, we integrated the ROS-responsive nanoparticles coated with the RBC membrane modified by the stroke homing peptide, SHp, aiming to develop a smart bioengineered drug delivery carrier (designated as SHp-RBCNP) as shown in Figure 1. The SHp-RBC-NPs were used to deliver a neuroprotective agent, NR2B9C, which can selectively disrupt the interaction of N-methyl-D-aspartate receptors (NMDARs) with the postsynaptic density protein (PSD-95)

RESULTS AND DISCUSSION Physicochemical Characterization. The boronic ester conjugated with dextran (PHB-dextran) as the ROS bioreponsive polymer was successfully synthesized and confirmed by 1H NMR (Supporting Information, Figure S1). It was indicated that the boronic ester was chemically conjugated onto hydroxyl groups of dextran. 1H NMR results demonstrated that the graft ratio of dextran coupled to boronic ester was 30.7% ± 2.1%. The 1H NMR was also used to confirm the chemical structure of maleinimide-PEG-DSPE (Mal-PEG-DSPE) and SHp-PEG-DSPE (Supporting Information, Figure S2). B

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Figure 2. Physicochemical characterization of nanoparticle formulations. (A) TEM images and hydrodynamic size distribution of RBC coated nanoparticles (a) and uncoated nanoparticles (b); scale bar = 200 nm. (B) Change in the particle size and ζ potential of nanoparticles after coating with RBC membrane (n = 3). (C) UV−vis spectra of NP and RBC-NP. (D) Digital photos demonstrating ROS-triggered hydrolysis of the nanoparticles in PBS (pH 7.4) and PBS with 1 mM H2O2. (E) In vitro release profiles of NR2B9C from NP and RBC-NP in PBS (pH 7.4) and PBS with 1 mM H2O2 (n = 3).

RBC-NPs were fabricated by coating the RBC membrane onto the surface of ROS-responsive nanoparticles via cosonication as shown in Figure 1. Transmission electron microscopy (TEM) was used to characterize the particle size and morphology of the plain nanoparticles (NPs) and RBCNPs. The RBC-NPs exhibited a distinctive spherical core−shell structure (Figure 2A). Moreover, the average hydrodynamic diameter of RBC-NPs was slightly increased to 194.6 ± 8.5 nm, and the ζ potential was decreased to −12.3 ± 2.1 mV, with respect to the NPs with an average diameter of 163.3 ± 4.6 nm and ζ potential of −21.1 ± 1.3 mV (Figure 2B). An outer layer was clearly observed in the TEM images, indicating the shell structure of the nanoparticles, which was also confirmed by the measurement of the difference in the particle size after coating by DLS (an outer lipid shell layer was found to be about 20−30 nm in thickness). The relatively increased particle size and decreased surface ζ potential suggested that a membrane was successfully translocated onto the surface of ROS-triggered nanoparticles. Additionally, the change in the UV absorption before and after the fusion process of the nanoparticles and RBC vesicles was observed (Figure 2C), showing that after coating with RBC membrane, a characteristic UV absorption profile was observed for RBC-NP (similar to that of RBCmembrane derived vesicles), as compared to those of the NP. The results of the other physicochemical properties charac-

terized for NPs and RBC-NPs are shown in the Supporting Information, Table S1. Hydrolysis profiles of the ROS-responsive nanoparticles were investigated in 0.01 M PBS (pH 7.4) and 1 mM H2O2 containing PBS. The nanoparticle solutions had light blue opalescence even after 4 h incubation in PBS, while they became completely clear at 30 min in the presence of H2O2 (Figure 2D). For quantitative study, the UV−vis spectra showed that 95% of nanoparticles were hydrolyzed in the medium containing 1 mM H2O2 within 30 min, while only 5% were found to be hydrolyzed in PBS in the absence of H2O2 (Supporting Information, Figure S3), indicating a functional ROS triggered sensitivity of nanoparticles made from PHBdextran. Similarly, hydrolysis profiles of the nanoparticles were also monitored in 0.01 M PBS containing 10% FBS and various concentrations of H2O2 at 37 °C by DLS (Supporting Information, Figure S3). The results of UV−vis spectra concerning the particle size indicated the great ROS triggered sensitivity of SHp-RBC-NP in FBS containing PBS. To test the physical stability of the RBC mimicking nanoparticles, the change in the particle size of the SHp-RBC-NP in PBS with and without 10% FBS was monitored over time by DLS. The SHpRBC-NPs exhibited a superior colloidal stability without obvious increase in the particle size over a period of 2 weeks (Supporting Information, Figure S4) at room temperature, C

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ACS Nano possibly attributed to the hydrophilic surface glycan layers in the erythrocyte membrane.7 However, there was some slight particle size changes at 37 °C during the 2 weeks (Supporting Information, Figure S5), which suggested that the nanocarrier formulation should be kept in cool condition. It is well-known that NMDARs can mediate not only ischemic brain damage but also the essential neuronal excitation. Therefore, research efforts should be made to achieve an effective treatment of ischemic stroke without blocking the NMDARs. To the best of our knowledge, very few reports about nanocarriers containing NMDAR selective blocking agents have been published. NR2B9C is the nine COOH-terminal residues of NR2B (Lys-Leu-Ser-Ser-IleGluSer-Asp-Val), which has the potential to act as an anti-ischemic stroke acid peptide that can selectively disrupt the interaction of NMDARs with the postsynaptic density protein (PSD-95) to prevent the overproduction of nitric oxide (toxic signaling agent) without blocking the normal NMDAR functions (synaptic activity or calcium influx).15,16 However, NR2B9C was found to have difficultly crossing the blood−brain barrier (BBB) and targeting to ischemic neurons. Then, an attempt was made to modify NR2B9C with Tat from HIV, but the safety of Tat was controversial. In this study, NR2B9C was loaded into a bioengineered, targeted, ROS-responsive smart nanocarrier to protect neurons from excitotoxicity in ischemic stroke. The in vitro release profile of nanoparticles loaded with rhodamine labeled NR2B9C was evaluated in release medium with or without 1 mM H2O2 to determine the oxidation effect on the release rate of the nanoparticle system (Figure 2E). Results showed the cumulative NR2B9C release was about 50% from NP, RBC-NP, and SHp-RBC-NP groups in the presence of 1 mM H2O2. However, there was only about 10% NR2B9C release in the absence of H2O2. This indicates that release rate from NPs, RBC-NPs, and SHp-RBC-NPs increased markedly in the presence of 1 mM H2O2 as compared to the non-H2O2 treatment groups. It was found that there was a negligible difference in the release profile among different nanoparticle formulations. The Transportation of Nanoparticles across BBB in Vitro. To investigate the ability of the various formulations across in vitro BBB, the free NR2B9C, NP/NR2B9C, RBC-NP/ NR2B9C, and SHp-RBC-NP/NR2B9C were added into the apical chamber, and the concentrations of NR2B9C in the basal chamber were measured. These results showed that the ratio of NR2B9C across the BCEC monolayer for 2 h was about 0.53%, 1.44%, 1.71%, and 1.67% for the free NR2B9C group, NP/ NR2B9C group, RBC-NP/NR2B9C group, and SHp-NP/ NR2B9C group, respectively (Figure 3A). It is suggested that nanoparticle formulations could enhance the NR2B9C transport across the in vitro BBB in comparison to the free NR2B9C. Importantly, the modification with SHp did not significantly increase BBB penetration as compared with the unmodified group, suggesting that the target binding site of SHp might be not in the brain capillaries but in the injured neurons in the ischemic stroke site.14 Therefore, it is indicated the SHp-RBCNPs in this study may enhance the BBB penetration of NR2B9C and target to damaged neurons in the ischemic region. Collectively, the bioengineered nanoparticle could be applied as a carrier of neuroprotective agents to home to ischemic stroke. Neuroprotective Effect of Nanoparticle Formulations. The in vitro cerebral ischemia therapeutic potential of the bioengineered nanoparticles was studied in this study. The

Figure 3. In vitro cell-based studies. (A) The ratio of NR2B9C loaded in various formulations transported across BBB in vitro. (B) Neuroprotective effect of various formulations after glutamate treatment in PC-12 cells. (Glu indicates glutamate and glycine at a concentration of 20 mM and 4 mM, respectively). The cells were treated with free NR2B9C, NP/NR2B9C, RBC-NP/NR2B9C, and SHp-RBC-NP/NR2B9C at a drug concentration of 800 ng/mL (equivalent to NR2B9C). (C) Quantitative data showing the intracellular ROS levels after exposure to various formulations. Statistical analysis used one-way ANOVA test. Differences were considered significant when *P < 0.05, **P < 0.01, or ***P < 0.001, respectively (n = 5).

detrimental cascades after cerebral ischemia are not clearly defined yet. However, it is generally recognized that the mass release of glutamate and a relatively high level of neurotoxic ROS from neurons play a significant role in neuronal cell injury and apoptosis.17,18 Glutamate and glycine induced PC-12-based cell model is widely used as a neurotoxic evaluation tool in D

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Figure 4. (A) In vivo pharmacokinetics of free NR2B9C, NP/NR2B9C, RBC-NP/NR2B9C, and SHp-RBC-NP/NR2B9C (n = 3). Error bars indicated SD. (B) Ex vivo fluorescent image of rhodamine-labeled free NR2B9C, NP, RBC-NP, and SHp-RBC-NP in the ischemic brain sections at 1, 2, and 6 h, respectively (n = 3). (C) Fluorescence ratio of ischemic hemisphere to the nonischemic hemisphere at 1, 2, and 6 h, respectively. Statistical analysis used one-way ANOVA test. Differences were considered significant when *P < 0.05, **P < 0.01, or ***P < 0.001, respectively.

vitro.19−23 The protective potential of different nanoparticle formulations is presented in Figure 3B. Results showed that the glutamate can reduce the cell viability to 60% without any treatment, while the viability of the cells increased to about 90% in the presence of free NR2B9C. As expected, the cell viability was significantly increased after exposure to SHp-RBC-NP/ NR2B9C, suggesting that the nano-biomimic carrier could achieve the protective effect on glutamate-induced cellular injury. Intracellular ROS Detection. DCFH-DA reactive oxygen species assay kit was used as a probe for intracellular ROS detection in this study. As shown in Figure 3C, it was found that the level of ROS induced by glutamate was increased to about 291% as compared with the control group cells, resulting in a high intracellular ROS microenvironment, which would

facilitate the degradation of the ROS stimuli-sensitive nanoparticles. Importantly, treatment with free NR2B9C and SHpRBC-NP/NR2B9C can significantly reduce the ROS level. Overall, the in vitro results suggested the potential of SHpRBC-NP/NR2B9C being used for the treatment of cerebral ischemia stroke. Interestingly, after treatment, it was found that the blank nanoparticles could reduce the ROS level to about 231%. Because ROS production is closely related to both cellular and tissue injuries induced by oxidative stress, this preliminary result suggested that certain nanoparticle formulations alone may be applied to relieve the pathological conditions by consuming excessive ROS, which was in agreement with recent reports that specific nanoparticles could function as scavengers of ROS.24,25 In fact, several ROS-scavenging plain nanoparticles have emerged as a useful E

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Figure 5. (A) Neuroscores of rats after cerebral ischemia. (B) Quantification of brain infarct volume at 24 h after MCAO in rats. (C) Representative TTC-stained brain sections of Sham-operated group, MCAO group, NP group, free NR2B9C group, NP/NR2B9C group, RBC-NP/NR2B9C group, and SHp-RBC-NP/NR2B9C group. The nonischemic region is observed as red, and the infarct region is shown in white. (D) Representative tissue slices showing that RBC-NP/NR2B9C and SHp-RBC-NP/NR2B9C group can significantly reduce the infarct volume; the arrows indicated the infarct region as observed. (E) Histochemistry analysis of brain, heart, liver, spleen, lung, and kidney tissue sections stained with hematoxylin−eosin; bar 100 μm. Data are expressed with mean ± SD (n = 7). Statistical analysis used one-way ANOVA test. Differences were considered significant when *P < 0.05, **P < 0.01, or ***P < 0.001, respectively.

RBC-NP/NR2B9C were found to be significantly higher than those of NP/NR2B9C without the membrane camouflage, indicating the stealth capability of the RBC membrane, confirming the successful coating of RBC membranes onto the nanoparticles, which plays an important role in the predominant in vivo mimicking properties of the smart bioengineered nanoparticles.5,32 Thus, the prolonged systemic circulation delivery might synergize with SHp peptide homing ability to improve the brain delivery of the therapeutic agent. Ex Vivo Distribution in MCAO Ischemic Rat Model. To evaluate the active targeting capability of the bioengineered nanoparticles to the ischemic site in the rat brain, the middle cerebral artery occlusion (MCAO) model rats were used for this study. The MCAO rats were intravenously injected with free NR2B9C, NPs, RBC-NPs, or SHp-RBC-NPs (containing equal dosages of rhodamine labeled NR2B9C), and ex vivo fluorescent imaging of the ischemic brains was conducted. As presented in Figure 4B, the fluorescent intensity observed in both RBC-NP and SHp-RBC-NP groups was much higher than that of NP and free NR2B9C groups as observed in the brain sections at 1, 2, and 6 h, suggesting that a higher amount of the therapeutic agent was accumulated into the brain sections via long circulation of RBC camouflage. It seems that the nonischemic hemisphere fluorescent intensity of the SHpRBC-NP group was higher than that of RBC-NP group from

pharmaceutical strategy to achieve specific neuroprotective effect.26,27 Similarly, vascular targeting of antioxidant nanomedicine may also provide wide medical applications such as management of oxidative stress and other toxicities.28−31 Pharmacokinetic Study. To evaluate the in vivo circulation behavior of different nanoparticle formulations, SD rats were used as the animal model. Briefly, the rats were intravenously administered different formulations at the same dose of rhodamine labeled NR2B9C. Then, blood samples were taken at specific time points, and the fluorescence intensity in the plasma was assessed for quantitation. As shown in Figure 4A, longer circulation time (over 48 h) was observed in both RBC-NP/NR2B9C and SHp-RBC-NP/NR2B9C groups as compared to the control group, probably due to the immuneevasion ability of the RBC membranes. At 24 and 48 h, it was found that there was still about 17% and 12% of the SHp-RBCNP/NR2B9C remaining in the blood circulation, while the plain nanoparticles were found to be rapidly eliminated (could not be detected after 24 h). It should be noted that free NR2B9C showed much lower retention in blood as expected with comparison to the other groups. In this study, a twocompartment PK model was used to calculate the PK parameters (Supporting Information, Table S2). In addition to the observed longer blood retention profile, the area under the curve (AUC) and the elimination half-life (t1/2) of SHpF

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METHODS

one single brain section such as the third section of 2 h. However, the accumulative fluorescence intensity of five brain sections of the SHp-RBC-NP group was not higher than that of RBC-NP group in the nonischemic hemisphere. Importantly, the fluorescence ratio of the ischemic hemisphere to the nonischemic hemisphere in SHp-RBC-NP group was much higher than that of RBC-NP group (Figure 4C), further demonstrating the selective distribution of the active targeting ability of the bioengineered nanoparticles to the ischemic site through chemical modification of the nanoparticles with SHp peptides. Collectively, in this study, it was demonstrated that SHp could actively improve the targeting capability of the nanoparticles to the ischemic neurons in brain, and the mechanism of the SHp homing to the ischemic site may be associated with the overactivation of glutamate receptors of the injured neurons at the ischemic stroke site.13,14,33 In Vivo Anti-Ischemic-Stroke Efficacy. The in vivo antiischemic stroke effect was assessed using the MCAO rat model. The neuroscore and infarct size were evaluated 24 h after the ischemic reperfusion. As shown in Figure 5A−D, no infarct and neurological deficiencies were found in the sham-operated rats. An obvious increase in the brain infarct size and neuroscores was observed in the MCAO rat group. More importantly, treatment with SHp-RBC-NP/NR2B9C significantly ameliorated neurological deficit induced by ischemia reperfusion as compared with that of the MCAO group (P < 0.001, Figure 5A−D), suggesting that nanoparticles functionalized with SHp and RBC membrane provided greater neuroprotective effect than the other groups. In Vivo Preliminary Safety Evaluation. To establish a preliminary in vivo safety profile of different types of nanoparticles, various nanoparticle formulations including NPs, RBC-NPs, and SHp-RBC-NPs were injected to the healthy mice via the tail vein. Then the serum biochemical analyses and histopathology evaluation were performed to monitor the potential toxicity. It was found that there were no significant abnormalities in the serum chemistry tests (AST, ALT, BUN, and creatinine levels) for renal and hepatic functionality analysis after daily administration for 1 week (Supporting Information, Table S3). H&E staining of the tissue samples including liver, heart, spleen, kidney, lung, and brain after the treatment with saline, plain NPs, RBC-NPs, and SHpRBC-NPs was conducted. Results showed that there was no evidence of abnormal and inflammatory cell infiltration in tissue sections (Figure 5E), demonstrating the good in vivo biocompatibility of the nanoparticle delivery system used in this study.

Materials. NR2B9C and cysteine modified SHp (CLEVSRKNC) were obtained from Shanghai GL Biochem Ltd. (Shanghai, China). Maleinimide-PEG-DSPE (Mal-PEG-DSPE) was purchased from Nanocs Inc. (New York, NY, USA). Fetal bovine serum (FBS), RPMI 1640 medium, and 0.25% (w/v) trypsin solution were purchased from Gibco BRL (Gaithersberg, MD, USA). DCFH-DA Assay Kit was purchased from Beyotime Biotechnology Co., Ltd. (Nantong, China). 4-(Hydroxymethyl) phenylboronic acid pinacol ester (PBAP), 4-dimethylaminopyridine (DMAP), and 1,1′-carbonyldiimidazole (CDI) were obtained from Sinoreagent Chemical Reagent Co., Ltd. (Shanghai, China). Cell Lines and Animals. PC-12 cells (rat adrenal pheochromocytoma cell line) and BCECs (rat brain capillary endothelial cell line) were obtained from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The cell lines were cultured in DMEM medium and RPMI 1640 medium, respectively, and supplemented with 10% (v/v) FBS, 1% penicillin, and 100 mg/mL streptomycin sulfate at 37 °C in 5% CO2 atmosphere. Sprague−Dawley (SD) rats (male, 5−6 weeks, 200 ± 20 g) and ICR mice (male, 4−5 weeks, 20 ± 2 g) were supplied by Shanghai SIPPR-Bk laboratory animal Co. Ltd. (Shanghai, China). All animal experiments were performed in accordance with guidelines approved by the Ethics Committee of Nanjing Medical University (Nanjing, China) and repeated in doubleblinded manner. Synthesis and Characterization of PHB-Dextran. PBAP (5.85 g, 25 mmol) was dissolved in anhydrous CH2Cl2 (30 mL) in a flamedried 100 mL flask. Carbonyldiimidazole (8.11 g, 50 mmol) was added and then stirred for 1 h. The mixture was washed with H2O (3 × 10 mL) first. Then the organic phase was washed with brine (1 × 10 mL), dried with MgSO4, and concentrated in vacuo to get a pure white solid (PBAP-CDI).34 Dextran (MW = 10000 g/mol, 45 mg, 0.0045 mmol) was put into to a flame-dried 25 mL flask and dissolved in anhydrous formamide (4 mL). Then DMAP (100 mg, 0.82 mmol) was added followed by the addition of CDI-activated PBAP-CDI (180 mg, 0.55 mmol). The reaction was performed overnight by magnetic stirring at 37 °C. The modified dextran was dialyzed using a dialysis bag (molecular weight cut off, 500 Da) in deionized (DI) water for 48 h and lyophilized to obtain a white solid PHB-dextran (0.148 g), which was further confirmed by 1H NMR. Preparation of ROS-Triggered Nanoparticles. The selfassembly formulation strategy was employed to prepare ROS-triggered nanoparticles. Briefly, PHB-dextran (10 mg) was dissolved in 0.5 mL of formamide and methanol (1:1, v/v). The resulting solution was added dropwise into 10 mL of Poloxamer 188 aqueous solution (0.5%, w/v) followed by magnetic stirring for 2 h at 37 °C. The solution was further dialyzed using dialysis bag (molecular weight cut off, 50 kDa) in DI water to remove the organic solvent residues. For NR2B9C loaded nanoparticles, NR2B9C was dissolved in 0.2 M Tris-HCl buffer and then added into the organic phase, followed by the similar operation procedures. Preparation of RBC-Membrane-Derived Vesicles and SHpInserted RBC Membrane. Hypotonic hemolysis method was used for the preparation of RBC-membrane-derived vesicles as previously published.24,25,35 Briefly, fresh whole blood was withdrawn from SD rats with low molecular heparin solution for anticoagulation. The density of RBCs collected was about 3 × 109 cells/mL. The whole blood was centrifuged (5000 rpm, 10 min) at 4 °C to separate the erythrocytes from the serum and leukocytic cream. The resultant erythrocytes were washed with PBS (pH 7.4) three times, and then 40 mL of 0.25× PBS was added into RBCs collected from 1 mL of blood for hemolysis, which was conducted at 4 °C for 2 h. After this hypotonic buffer treatment, the solution was centrifuged twice (9000 rpm, 15 min) to remove the released hemoglobin. The pink pellet was then resuspended in 1 × PBS at 4 °C. To form the SHp-inserted RBC membrane, the SHp-PEG-DSPE was synthesized via Michael addition reaction of Mal-PEG-DSPE and SHp. Then, the SHp-PEG-DSPE was

CONCLUSION A smart neuroprotective agent loaded bioengineered ROSresponsive nanoparticle formulation for the treatment of ischemia stroke was developed, aiming to prolong the in vivo circulation time and enhance the active targeting ability of the therapeutic agent. In vitro evaluation showed that SHp-RBCNPs have the potential to provide greater protective effect on glutamate-induced PC-12 cytotoxicity in a cell-based model. Furthermore, ex vivo fluorescent imaging study showed that SHp-RBC-NPs exhibited improved active homing functionalities. Importantly, the SHp-RBC-NPs can also greatly ameliorate neuroscores and infarct volume in response to the surgical MCAO injury. Therefore, it suggested that SHp-RBCNPs may be utilized as a potential formulation strategy to enhance the treatment of ischemic stroke in the clinic. G

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ACS Nano co-incubated with the light pink solution for 1 h at room temperature, and the resultant SHp-inserted RBC membranes were then washed with PBS (pH 7.4) to remove the uninserted SHp-PEG-DSPE.36,37 The RBC-membrane-derived vesicles were prepared by a probe sonication method reported previously.38 The hydrodynamic diameter of the RBC-membrane-derived vesicles was measured by dynamic light scattering (DLS). Preparation of RBC-Membrane-Camouflaged ROS-Responsive Nanoparticles (RBC-NP). One milliliter of ROS-responsive nanoparticles was mixed with 0.2 mL of RBC-membrane vesicles. Then the RBC-NP was fabricated by sonication of the mixture in a water bath sonicator (42 kHz) at 100 W for 5 min.39 Redundant vesicles were removed by centrifuging at 12000 rpm for 15 min at 4 °C.40 Physicochemical Characterization. The ζ potential and hydrodynamic diameter of different types of nanoparticles were measured by DLS (Zs90, Malvern, U.K.). The morphology of RBC-NPs was further observed by transmission electron microscope (TEM) (JEM-1230, Japan). UV−vis spectrometry (Synergy 2, BioTek Instruments Inc., USA) was also applied to characterize the NPs and RBC-NPs. Hydrolysis of the nanoparticles triggered by ROS was performed in 0.01 M PBS and PBS containing various concentrations of H2O2 at 37 °C. Digital photos during the hydrolysis process were taken for direct illustration. Quantitative experiments were conducted by the measurement of the UV absorbance of nanoparticles at 280 nm at predetermined time intervals.24 The physical stability testing of SHpRBC-NP in PBS with and without 10% FBS was monitored by DLS. The in vitro release testing of NPs and RBC-NPs (containing 100 μg/ mL of NR2B9C labeled with rhodamine) was performed by suspending different nanoparticle formulations in PBS (pH 7.4) with or without 1 mM H2O2, respectively. At the predetermined time intervals, the fluorescence intensity of NR2B9C labeled with rhodamine was measured by a fluorescence spectrometer (F-4600, Hitachi, Japan) at 525 nm with an excitation wavelength of 460 nm. Transport across the BBB: In Vitro BBB Model. The in vitro BBB model was built as described.14 Briefly, BCECs were cultured on transwell filters for 3 days. Once the cells were 90% confluent, they were serum-starved for another 3 days with 103 nmol/L of hydrocortisone. Then the cells are ready for BBB transport studies after measuring transepithelial electrical resistance (TEER) values. The free NR2B9C, NP/NR2B9C, RBC-NP/NR2B9C, and SHp-RBC-NP/ NR2B9C (equivalent to 800 ng/mL NR2B9C) were added to the apical chamber of the models to screen the transport profile. After 2 h of treatment, a sample with a volume of 800 μL was taken from the basal chamber. The concentration of NR2B9C in the basal chamber was determined using fluorescence spectrophotometer (F-4600, Hitachi, Japan) at 525 nm with an excitation wavelength of 460 nm. Neuroprotective Effect of Nanoparticle Formulations. PC-12 cells were cultured in RPMI 1640 medium containing 10% (v/v) FBS, 1% penicillin, and 100 mg/mL streptomycin sulfate in a 5% CO2 at 37 °C. Cells were seeded in 96-well plates in 100 μL of RPMI 1640 medium to acquire a concentration of 1 × 104 cells/well. After 24 h incubation, glutamate (20 mM) and glycine (4 mM) were incubated with PC-12 cells as previously reported,41 followed by addition of blank NP, free NR2B9C, NP/NR2B9C, RBC-NP/NR2B9C, and SHpRBC-NP/NR2B9C (equivalent to 800 ng/mL NR2B9C). After incubation at 37 °C for 24 h, the cell viability was tested using MTT assay. Intracellular ROS Detection. The generation of intracellular ROS was monitored using DCFH-DA assay kit as published previously.42 PC-12 cells were seeded in 24-well plates at a concentration of 1 × 105 cells/well and incubated for 24 h. Then, cells were treated with glutamate (20 mM) and glycine (4 mM), followed by addition of blank NP, free NR2B9C, NP/NR2B9C, RBC-NP/NR2B9C, and SHpRBC-NP/NR2B9C (equivalent to 800 ng/mL NR2B9C). After incubation at 37 °C for 24 h, cells were washed and treated with 10 μM DCFH-DA for 60 min in the dark at 37 °C. The supernatant was removed, and the cells were washed with cold PBS three times. The fluorescence intensity of relative ROS units was measured by Tecan

infinete M-200 microplate reader (excitation 488 nm, emission 525 nm). Pharmacokinetic Study. In vivo pharmacokinetic testing of different formulations was carried out using SD rat model. The rats were divided into different groups and intravenously injected with free NR2B9C, NP, RBC-NP, and SHp-RBC-NP (equivalent to 800 ng/mL NR2B9C labeled with rhodamine). At pre-established time points (5, 10, 15, and 30 min and 1, 2, 4, 8, 24, and 48 h), blood samples were taken and centrifuged at 5000 rpm for 10 min. The supernatant was collected, and the fluorescence intensity of NR2B9C labeled with rhodamine was measured. The two-compartment model was fitted for pharmacokinetic parameters as reported.5 Ex Vivo Testing in MCAO Ischemic Rat Model. The improved middle cerebral artery occlusion (MCAO) method was used to induce focal cerebral ischemia and reperfusion as established previously.43 In brief, male SD rats were anesthetized using 10% chloral hydrate by intraperitoneal injection. The rectal temperature was kept at about 36 °C using a heating blanket during surgery. After a median incision of the neck skin, the common carotid artery (CCA) and the internal carotid artery (ICA) were separated and ligated transiently with careful conservation of the vagal nerve. The external carotid artery (ECA) was also separated, and then a monofilament nylon suture coated with silicon was inserted from the ECA to ICA to block the origin of MCA. After 2 h, reperfusion was initiated by withdrawing the monofilament.43 Then the MCAO model rats were intravenously administered free NR2B9C, NP, RBC-NP, or SHp-RBC-NP (equivalent to 100 μg/ mL NR2B9C labeled with rhodamine) via the tail vein. The distribution of rhodamine labeled NR2B9C in the brain was detected at 1, 2, and 6 h, specifically. Then the brain tissues were taken rapidly and frozen at −20 °C for 5 min. Brain coronal slices were made at 2 mm thick, followed by washing steps with saline. Meanwhile, other major organs (heart, liver, spleen, lung, and kidney) at 1, 2, and 6 h were also harvested and washed with saline. These organs and the brain sample slices were imaged by bioluminescence imaging system (IVIS Spectrum System, Caliper Life Sciences, USA) equipped with an excitation wavelength at 550 nm and an emission wavelength at 590 nm. Exposure time was set as 30 s per image.44 In Vivo Anti-Ischemic-Stroke Efficacy. For the in vivo antiischemic stroke efficacy testing, SD rats were weighed and randomly divided into seven groups (n = 7). Sham-operated group was used as the negative control, and the MCAO model rats were intravenously injected with saline, blank NP, free NR2B9C, NP/NR2B9C, RBCNP/NR2B9C, or SHp-RBC-NP/NR2B9C at a concentration of 0.3 mg/kg. The neuroscore evaluation and infarct size measurement were conducted at 24 h after the MCAO surgery. Neuroscore assessment was performed using a well-established five point scale methodology (Rating scale, 4 = spontaneous circling, 3 = circling to left by pulling the tail, 2 = decreased grip strength of left forepaw, 1 = failure to extent left forepaw, and 0 = no deficit).43 After the neuroscore assessment, the rats were sacrificed, and brain tissues were removed immediately and frozen at −20 °C for 5 min. The brain slices were made at 2 mm thick and then stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37 °C for 30 min. The tissue sections were stored in 4% paraformaldehyde solution. The TTC-stained sections were photographed, and a percentage area of the coronal section in the infarcted hemisphere was shown as the infarct volume using image tool 3.0.43 In Vivo Preliminary Safety Evaluation. Twenty male ICR mice were randomly separated into four groups (n = 5): the saline group, the NP group, the RBC-NP group, and the SHp-RBC-NP group. Different nanoparticle formulations (100 mg/kg) and saline were intravenously administered daily for 7 days. The body weight was monitored daily after each administration. Major organ tissues (brain, heart, liver, kidney, spleen, and lung) and blood samples were collected at 24 h after the seventh administration for the hematological analysis using Hitachi 7080 Chemistry Analyzer (Hitachi Ltd., Japan). The organ samples were fixed using paraformaldehyde for 48 h. Then the tissues were sectioned at a thickness of 5 μm and stained with hematoxylin and eosin (H&E) followed by visualization under the fluorescent microscope. The urea nitrogen (BUN), creatinine, serum H

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ACS Nano aspartate transaminase (AST), and alanine transaminase (ALT) levels were evaluated using Hitachi 7080 Chemistry Analyzer (Hitachi Ltd., Japan). Statistical Analysis. All the results were expressed as mean ± standard deviation (SD). Statistical analysis was performed with SPSS 20.0 software. Statistical analysis was performed using one-way ANOVA test. Differences were considered significant when *P < 0.05, **P < 0.01, or ***P < 0.001.

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.8b00477. Scheme of the synthesis and degradation of PHBdextran, synthesis of SHp-PEG-DSPE, 1H NMR spectra of PHB-dextran, Mal-PEG-DSPE, and SHp-PEG-DSPE, ROS-triggered hydrolysis curves and particle size changes of SHp-RBC-NP at various concentrations of H2O2, TEM images of SHp-RBC-NP/NR2B9C treated with 1 mM H2O2 for 6 h, stability of SHp-RBC-NP at 25 and 37 °C, in vitro cytotoxicity of different nanoparticles against BCECs, the biodistribution of various nanoparticles in major organs, characterizations of NPs, pharmacokinetic parameters of free NR2B9C, NP, RBC-NP, and SHpRBC-NP, and mouse serum level of biochemical variables after intravenous treatment with saline, NP, RBC-NP, or SHp-RBC-NP (PDF)

AUTHOR INFORMATION Corresponding Author

*H.X. E-mail: [email protected]. ORCID

Hongliang Xin: 0000-0002-2966-7381 Author Contributions #

W.L. and J. X. contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was kindly supported by National Natural Science Foundation of China (31671018), Natural Science Foundation of Jiangsu Province-Excellent Young Scientist Fund (BK20160096), and Six Talent Peaks Project of Jiangsu Province (SWYY-051). REFERENCES (1) Panagiotou, S.; Saha, S. Therapeutic Benefits of Nanoparticles in Stroke. Front. Neurosci. 2015, 9, 182. (2) Lee, D.; Bae, S.; Hong, D.; Lim, H.; Yoon, J. H.; Hwang, O.; Park, S.; Ke, Q.; Khang, G.; Kang, P. M. H2O2-Responsive Molecularly Engineered Polymer Nanoparticles as Ischemia/Reperfusion-Targeted Nanotherapeutic Agents. Sci. Rep. 2013, 3, 2233. (3) Broaders, K. E.; Grandhe, S.; Fréchet, J. M. J. A Biocompatible Oxidation-Triggered Carrier Polymer with Potential in Therapeutics. J. Am. Chem. Soc. 2011, 133, 756−758. (4) Lippert, A. R.; Van de Bittner, G. C.; Chang, C. J. Boronate Oxidation as a Bioorthogonal Reaction Approach for Studying the Chemistry of Hydrogen Peroxide in Living Systems. Acc. Chem. Res. 2011, 44, 793−804. (5) Hu, C. M.; Zhang, L.; Aryal, S.; Cheung, C.; Fang, R. H.; Zhang, L. Erythrocyte Membrane-Camouflaged Polymeric Nanoparticles as a Biomimetic Delivery Platform. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 10980−10985. I

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