Dual targeted nanocarrier for brain ischemic stroke treatment

    Dual targeted nanocarrier for brain ischemic stroke treatment Yue Zhao, Yan Jiang, Wei Lv, Zhongyuan Wang, Lingyan Lv, Baoyan Wang, Xin Liu, Yang Liu, Quanyin Hu, Wujin Sun, Qunwei Xu, Hongliang Xin, Zhen Gu PII: DOI: Reference:

S0168-3659(16)30252-8 doi: 10.1016/j.jconrel.2016.04.038 COREL 8241

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

21 February 2016 23 April 2016 28 April 2016

Please cite this article as: Yue Zhao, Yan Jiang, Wei Lv, Zhongyuan Wang, Lingyan Lv, Baoyan Wang, Xin Liu, Yang Liu, Quanyin Hu, Wujin Sun, Qunwei Xu, Hongliang Xin, Zhen Gu, Dual targeted nanocarrier for brain ischemic stroke treatment, Journal of Controlled Release (2016), doi: 10.1016/j.jconrel.2016.04.038

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Dual targeted nanocarrier for brain ischemic stroke treatment

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Yue Zhao1#, Yan Jiang1#, Wei Lv1, Zhongyuan Wang1, Lingyan Lv1, Baoyan Wang1, Xin Liu1, Yang Liu1, Quanyin Hu2, 3, Wujin Sun2, 3, Qunwei Xu1*, Hongliang Xin1*,

These authors contributed equally to this manuscript.

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Zhen Gu2, 3, 4

Nanjing 211166, China

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1. Department of Pharmaceutics, School of Pharmacy, Nanjing Medical University,

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2. Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, USA

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3. Division of Molecular Pharmaceutics and Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA 4. Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA

Corresponding author: Qunwei Xu(Tel.: +86-25-86868468, Fax: +86-25-86868467, E-mail addresses: [email protected]); Hongliang Xin(Tel.: +86-25-86868476, Fax: +86-25-86868467, E-mail addresses: [email protected]). 1

ACCEPTED MANUSCRIPT ABSTRACT Focal cerebral ischemia, known as stroke, causes serious long-term disabilities

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globally. Effective therapy for cerebral ischemia demands a carrier that can penetrate

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the blood-brain barrier (BBB) and subsequently target the ischemia area in brain. Here, we designed a novel neuroprotectant (ZL006) loaded dual targeted nanocarrier based on liposome (T7&SHp-P-LPs/ZL006) conjugated with T7 peptide (T7) and

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stroke homing peptide (SHp) for penetrating BBB and targeting ischemia area,

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respectively. Compared with non-targeting liposomes, T7&SHp-P-LPs/ZL006 could transport across BCEC cells and significantly enhance cellular uptake and reduce cells

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apoptosis of excitatory amino acid stimulated PC-12 cells. However, there was no

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significant difference in cellular uptake between SHp-modified and plain liposomes

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when PC-12 cells were incubated without excitatory amino acid. Besides, ex vivo fluorescent images indicated that DiR labeled T7&SHp-P-LPs could efficiently

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transport across BBB and mostly accumulated in ischemic region rather than normal cerebral hemisphere of MCAO rats. Furthermore, T7&SHp-P-LPs/ZL006 could enhance the ability of in vivo anti-ischemic stroke of MCAO rats. These results demonstrated that T7&SHp-P-LPs could be used as a safe and effective dual targeted nanocarrier for ischemic stroke treatment.

Keywords: Stroke; Stroke homing peptide; Dual-targeting Liposomes; ZL006; Neuroprotection

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ACCEPTED MANUSCRIPT 1. Introduction Ischemic stroke leads to severe morbidity and a high mortality, which is

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recognized as one of the most serious public health problems worldwide[1]. Although

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different mechanisms are involved in the pathogenesis of stroke, there are increasing evidences showing the critical role of glutamate excitotoxicity, excessive stimulation of N-methyl-D-aspartate receptors (NMDARs), neuronal nitric oxide synthase (nNOS)

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activation, oxidative stress, and inflammation in the progression of ischemic brain

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injury precipitate the neuronal death and neurological dysfunction[2,3]. Activation of the NMDAR leads to Ca2+ influx as well as the regulation of signaling pathways

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including nNOS. Activation of nNOS via NMDAR requires interaction with the

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scaffold protein postsynaptic density 95 kDa (PSD-95), which forms an

interaction

complex.

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NMDAR/PSD-95/nNOS can

indirectly

inhibit

Therefore, the

disrupting

activity

of

the

NMDAR

nNOS-PSD-95 and

prevent

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glutamate-induced excitotoxicity[4,5]. ZL006, screened by our center to block the ischemia induced nNOS-PSD-95 association selectively, had potent neuroprotective activity in vitro and ameliorated focal cerebral ischemic damages in mice and rats that were subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Moreover, it had no effect on aggressive behavior or spatial memory[6]. However, the failure of chemotherapy is due to the inability of intravenously administered anti-ischemic agents to cross BBB and reach ischemic lesion. The effective therapeutics have less than optimal usefulness for ischemic stroke, mainly owing to the low concentration of therapeutic 3

ACCEPTED MANUSCRIPT agents accumulated on ischemic lesion and nonspecific diffusion into healthy tissue. Thus, there is an urgent need for novel drug delivery system capable of both

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penetrating BBB and targeting to ischemic lesion for ischemic stroke treatment.

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Dual-targeted liposome could transport more drugs across BBB and selectively deliver drugs to the ischemic tissue, enhancing the local concentration of the drugs while reducing their side effects.

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Receptor-mediated endocytosis is one of the major mechanisms of brain

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targeting drug delivery system[7]. A special ligand peptide of the over-expressed transferrin receptor (TfR) on the brain capillaries endothelial cells, HAIYPRH (T7),

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can specially bind to TfR and mediate the transport of nanocarriers across the

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BBB[8,9]. Recently, T7-conjuagted nanoparticles were reported for nanoscale

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brain-targeted magnetic resonance imaging[10] and with good targeting ability to brain tumors[11,12]. Meanwhile, in our previous study, we have confirmed that

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T7-modified liposomes could target to the brain, showing the potential as a targeted drug delivery system for ischemic stroke treatment. After improving drug accumulation in the brain, targeting ischemia area after penetrating BBB remains challenging. Hai-Yan Hong et al. found the CLEVSRKNC peptide as a stroke-homing peptide (SHp) by in vivo phage display in a rat model with focal cerebral ischemia. The SHp preferentially homed to ischemic brain tissue with a lack of distribution into non-ischemic tissue, it co-localized to a portion of neuronal cells undergoing apoptosis at the penumbra region of ischemic brain tissue but not to astrocytes therein 4

ACCEPTED MANUSCRIPT [13]. Thus, in our study, SHp was used to further improve drug accumulation in ischemia area. As shown in Fig. 1, the liposomes could transport ZL006 across the

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BBB (Grade I targeting) and then target ischemia area (Grade II targeting).

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Fig.1 Design of ZL006 loaded T7 and SHp conjugated PEGylated dual targeted nanocarrier for focal cerebral ischemia treatment via transferrin receptor mediated transcytosis and glutamate receptor mediated endocytosis.

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Nanoparticulate drug delivery systems have attracted increasing attention in recent years because of sustained release profile, high drug loading capacity and the property of passive targeting by the enhanced permeability to pathological lesion[14]. In this study, we designed T7 and SHp dual-conjugated PEGylated liposome (T7&SHp-P-LPs) as a dual targeted nanocarrier of ZL006 to enhance the anti-ischemic stroke efficacy.

2. Materials and methods 2.1. Materials ZL006 was kindly provided by Prof. F Li (Department of Medicinal Chemistry, 5

ACCEPTED MANUSCRIPT School of Pharmacy, Nanjing Medical University, Nanjing, China). Modified 7-amino acid peptide (HAIYPRH, T7) and 9-amino acid peptide (CLEVSRKNC, stroke

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homing peptide, SHp) were purchased from Shanghai GL Biochem Ltd (Shanghai,

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China). Mal-PEG-DSPE was purchased from Laysan Bio Co., USA. RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS) and trypsin solution were purchased from Gibco BRL (Gaithersberg, MD, USA). BCA kit and TritonX-100 was

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purchased from Beyotime biotechnology Co., Ltd. (Nantong, China). The other

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chemical reagents were of analytical grade and used as received. 2.2. Animals and cell line

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Spraguee-Dawley (SD) rats (male, 5-6weeks, 200±20 g) and ICR mice (male,

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4-5weeks, 20±2 g) were supplied by Animal Experimental Center of Nanjing Medical

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University (Nanjing, China). All animal experiments were performed in accordance with guidelines evaluated and approved by the ethics committee of Nanjing Medical

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University (Nanjing, China). BCEC cells (rat’s brain capillary endothelial cell line) and PC-12 cells (rat’s adrenal pheochromocytoma 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, 100U/mL penicillin and 100U/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO2. 2.3. Preparation of T7&SHp-P-LPs 6

ACCEPTED MANUSCRIPT The procedures of synthesis of T7-PEG-DSPE and SHp-PEG-DSPE were shown in supporting information. The liposomes, containing soybean lecithin, cholesterol,

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and DSPE-PEG2000 with molar ratio of 80/20/8, were prepared through the ethanol

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injection method as previously described[15,16]. In brief, lipids and T7-PEG-DSPE (2.5mg) were dissolved in anhydrous ethanol (6.5 mL) and SHp-PEG-DSPE (2.5mg) was dissolved in methanol (1 mL). The mixture of above was gently dripped into

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constantly stirring PB solution (pH 7.4, 50 °C). The resulting solution was sonicated

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using a probe sonicator (333w, work/stop: 2s/3s, 5 min) to decrease particle size. After further removal of the residual ethanol and methanol with a rotary evaporator (0.1

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MPa, 5 min, 60 °C), the liposome was extruded through polycarbonate membranes

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with the pore size ranging from 450 nm down to 220 nm. All the process was

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conducted in darkness. For ZL006 or DiR-loaded liposomes, both ZL006 and DiR were dissolved in anhydrous ethanol as the lipid part. The single peptide (T7- or SHp-)

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modified liposomes were prepared with the same procedure as mentioned above. 2.4. Characterization of T7&SHp-P-LPs. The morphology various liposomes were determined by transmission electronic microscopy (JEOL USA, Wilmington, DE, USA). The various liposomes were diluted with an appropriate volume of distilled water before measurement. 2.5. In vitro release The rate of in vitro release behavior of ZL006 from liposomes was determined using a dynamic dialysis method in phosphate-buffered saline solution (PBS, pH 7.4 and pH 5.5) containing 0.5% (w/v) Tween-80 at 37 °C[17]. Briefly, ZL006 loaded 7

ACCEPTED MANUSCRIPT T7&SHp-P-LPs (containing 800µg of ZL006) were dispersed in 2mL of mediator solution and sealed in dialysis bag (MWCO 3500). The liposome solution was then

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dialyzed against 50mL of PBS at 37 °C using a thermostatic shaker at an appropriate

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speed. A portion of 0.5 mL dialysate was taken out at various time points and replenished with 0.5 mL fresh buffer solution. The concentration of ZL006 released from liposomes was determined using HPLC with correction for the volume

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replacement.

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2.6. Transport across the BBB model in vitro

BBB model in vitro was built as described[18]. Briefly, BCEC cells were

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cultured on transwell filters for 3 days. Once the cells were 90% confluent, they were

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serum-starved for another 3 days with 103nmol/L of hydrocortisone, which increases

after

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the tight junction protein occludin[19]. The cells are ready for BBB transport studies measuring

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values.

Then,

P-LPs/ZL006,

T7-P-LPs/ZL006,

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SHp-P-LPs/ZL006 and T7&SHp-P-LPs/ZL006 at concentration of 400 μg/mL were added to apical chamber of the models to screen the transport profile, respectively. After 2 hour of treatment, sample with a volume of 900 µl was taken from the basal chamber. ZL006 content in the basal chamber was determined using HPLC. 2.7. Cellular uptake of T7&SHp-P-LPs/ZL006 by glutamate stimulated PC-12 cells PC-12 cells were seeded in 24-well plates at a density of 1×105 cells/well. After incubation for 24h and washed with PBS, PC-12 cells were incubated with glutamate (20mM) and glycine (4mM) for 2h at 37 °C as previously reported[20]. The medium 8

ACCEPTED MANUSCRIPT in each well was then incubated with free ZL006, P-LPs/ZL006, T7-P-LPs/ZL006, SHp-P-LPs/ZL006 and T7&SHp-P-LPs/ZL006 at different concentration (100, 200,

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300, 400µg/mL) for 2h at 37 °C, respectively. In a separate experiment to study the

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stroke homing function of SHp, PC-12 cells were incubated with normal culture medium without glutamate and added with P-LPs/ZL006 and SHp-P-LPs/ZL006, respectively.

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At the end of incubation, samples were collected and the cells were washed with

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cold PBS. The cells were lysed with PBS containing 1% TritonX-100 per well for 10 min. To investigate the total cell protein content, an aliquot of the cell lysate was used

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HPLC with UV detector[21].

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for BCA protein assay. The ZL006 concentration of the cell lysates was measured by

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2.8. Ex vivo fluorescent image of MCAO ischemic brain The focal cerebral ischemia was induced by the middle cerebral artery occlusion

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(MCAO) method as described previously[22]. In brief, male SD rats were anesthetized with 10% chloral hydrate. The common carotid artery (CCA) and the internal carotid artery (ICA) were isolated and ligated transiently. The external carotid artery (ECA) was isolated after a median incision of the median neck skin. After that a MCAO monofilament was inserted from the ECA to ICA to occlude the origin of MCA. After 2 h, the suture was removed to allow reperfusion of the ischemic area via common carotid artery[22]. Then, DiR labeled P-LPs, T7-P-LPs, SHp-P-LPs and T7&SHp-P-LPs were injected into MCAO model rats through the tail vein, respectively. The localization of the different liposomes in the brain was determined 6 9

ACCEPTED MANUSCRIPT h and 24 h after MCAO, respectively. After washing with saline, the brain sections were viewed by bioluminescence imaging system (IVIS Spectrum System, Caliper

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Life Sciences, USA)[23]. Image tool was used for statistical analysis.

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2.9. In vivo anti-ischemic stroke

In the present study, Sham-operated group was taken as negative control and MCAO model rats were injected with saline, free ZL006, P-LPs/ZL006,

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T7-P-LPs/ZL006, SHp-P-LPs/ZL006 and T7&SHp-P-LPs/ZL006 at the concentration

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of 4mg/kg, respectively.

Neurological deficit scores were evaluated by an examiner blinded to the

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experimental groups at 24 h after MCAO. The neurological deficit score was assessed

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using an established five point scale[22], which consists of grade 0: no neurological

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defect; grade 1: failure to fully extend the right forepaw; grade 2: circling to the right; grade 3: circling to the reverse of the right-the left; grade 4: rats did not walk

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spontaneously and had a depressed level of consciousness. The rats were euthanized and brains were harvested at 24 h after MCAO and neurological assessment. The brains were quickly removed and chilled in cold saline for 5 min followed by sectioning into 2 mm-thick coronal slices and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) at 37 °C for 30 min. Then the slices were immersed in 4% paraformaldehyde (PFA) solution for preservation[24]. TTC-stained sections were photographed and the digital images were analyzed using image tool 3.0. The percentage of infarction (infarct ratio) was calculated from the data of infarct volume and total coronal section using following formula[22]: 10

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2.10. TUNEL assay

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Coronal cryostat sections were processed according to the manufacturer’s

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instructions for TUNEL assay (Roche Systems, Inc., Basel, Switzerland). Briefly, the sections were incubated with proteinase K and 0.3% H2O2 at 25 °C for 1 h, and then

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treated with terminal deoxynucleotidyl transferase (TdT) at 37 °C for 1 h. 3,3'-diaminobenzidine (DAB) was used for the visualization of the apoptotic cells

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after further incubation with a peroxidase conjugated antibody. TUNEL-positive cells

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displayed a brown staining within the nucleus of the apoptotic cells. The penumbra of

microscope[24].

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each ischemic lesion was observed and the photographs were taken by a fluorescence

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2.11. Statistical analysis

All the results were expressed as mean ± standard deviation (SD). Statistical

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analysis was performed with SPSS 20.0 software. Statistical analysis was used one-way ANOVA test. Differences were considered significant when *P<0.05, **P<0.01, ***P<0.001, respectively. 3. Results and discussion 3.1. Characterization of SHp-PEG-DSPE The copolymers of T7-PEG-DSPE had been synthesized and characterized in our previous report[15]. The 1H NMR spectra of Mal-PEG-DSPE and SHp-PEG-DSPE were confirmed (Figure S1, Supporting Information). The maleimide group has a characteristic peak at 6.7 ppm in Mal-PEG-DSPE (Figure S1 a, Supporting 11

ACCEPTED MANUSCRIPT Information). The characteristic peak of methylene protons of DSPE and PEG was at 1.2 ppm and 3.7 ppm, respectively. However, the maleimide peak disappeared in the H NMR spectra of SHp-PEG-DSPE (Figure S1 b, Supporting Information) whereas

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conjugated with Mal-PEG-DSPE copolymer.

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the DSPE and PEG segment were still presented. It indicated that the SHp was

The SHp-PEG-DSPE copolymer was confirmed by FTIR analysis (Figure S2,

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Supporting Information). The spectrum of Mal-PEG-DSPE (Figure S2 a, Supporting

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Information) showed a broad N-H stretch band at 3600 – 3200 cm−1, centered at 3400 cm−1 and a weak C=O stretching vibrational absorption at 1686 cm−1. In the spectrum

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of SHp-PEG-DSPE (Figure S2 b, Supporting Information), due to the existence of

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more secondary amide groups in the structure of SHp, the intensity of the two bands

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remarkably increased. These results demonstrated the successful synthesis of SHp-PEG-DSPE.

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3.2. Characterization of liposomes Liposome can provide a versatile drug delivery vehicle owing to their excellent biocompatibility and special structure[25]. However, plain liposomes are lack of brain active targeting capacity. Therefore, active targeting liposomal delivery system based on receptor mediated endocytosis has been extensively investigated for brain disease therapies in recent years[26, 27]. In our study, liposomes were prepared by ethanol injection method. Due to the property of SHp-PEG-DSPE, we added moderate methanol to dissolve SHp-PEG-DSPE and then mixed with soya lecithin, cholesterol and ethanol. Methanol and ethanol were evaporated with a vacuum rotary evaporator 12

ACCEPTED MANUSCRIPT so that they would not cause extra toxicity. Besides, it was identified that the extra methanol did not caused the change in physical-chemical properties of liposomes.

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The characterizations of liposomes including particle size, polydispersity indexes

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(PDI), zeta potential, encapsulation efficiency (EE) and loading capacity (LC) were shown (Table S1, Supporting Information). The mean particle size of P-LPs and T7&SHp-P-LPs were 90.76±1.21nm and 96.24±1.13nm, respectively, and both of the

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liposomes exhibited a decent PDI. Meanwhile, the LC and EE of two kinds of ZL006

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loaded liposomes were very close, indicating that the incorporation of T7-PEG-DSPE and SHp-PEG-DSPE into liposomes had no influence on the physical properties of

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liposomes. The representative TEM images of these liposomes were shown in Fig. 2A,

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T7&SHp-P-LPs.

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B. There were no significant difference in morphology between P-LPs and

The accumulative release curves of ZL006 loaded T7&SHp-P-LPs under

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different pH conditions displayed similar release behaviors. After 48 h of incubation, the cumulative releases of ZL006 reached 66.8±1.9% and 70.3±5.8% under pH 5.5 and pH 7.4, respectively (Fig. 2C).

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Fig.2

TEM

image,

particle

size

and

size

distribution

of

P-LPs/ZL006

(A)

and

T7&SHp-P-LPs/ZL006 (B). ZL006 release profiles from T7&SHp-P-LPs in PBS (pH 5.5) and PBS (pH 7.4) containing 0.1% Tween-80 (n=3) (C).

3.3. The transportation of T7&SHp-P-LPs/ZL006 across BBB in vitro To investigate the ability of dual-targeting liposomes across BBB, P-LPs/ZL006, SHp-P-LPs/ZL006, T7-P-LPs/ZL006 and T7&SHp-P-LPs/ZL006 were added into the apical chamber and concentrations of ZL006 in the basal chamber were measured (Fig. 14

ACCEPTED MANUSCRIPT 3A). Among these liposome formulations, the T7 modified and dual-targeting liposome

exhibited

the

highest

BBB

penetrating

capability.

Therefore,

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T7&SHp-P-LPs could be used as a carrier to transport the drug across BBB.

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3.4. Cellular uptake of T7&SHp-P-LPs/ZL006 by glutamate stimulated PC-12 PC-12 cells were generally used to model the neuronal system[28, 29]. The cell model was widely applied for studying cellular glutamate toxicity, which was an

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important mechanism of neuronal apoptosis in cerebral ischemia[30, 31, 32]. PC-12

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cells were incubated with glutamate and glycine (5:1, Mol: Mol) for 1 h at 37 °C. The concentration of 20mM of glutamate led to about 50% inhibition of cell viability, thus

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this concentration of glutamate and glycine (20mM and 4mM, respectively) was

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chosen for the following study.

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Morphologies of PC-12 cells treated with different formulations were shown (Figure S3, Supporting Information). T7&SHp-P-LPs/ZL006 displayed obvious

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neuroprotective effect. The quantitative cellular uptake of free ZL006, P-LPs/ZL006, T7-P-LPs/ZL006, SHp-P-LPs/ZL006 and T7&SHp-P-LPs/ZL006 at different concentrations by glutamate stimulated PC-12 cells was presented in Fig. 3B. It showed that the uptake of liposomes was concentration-dependent. The uptake of T7&SHp-P-LPs/ZL006 was 1.25 and 1.3 -fold higher than that of P-LPS/ZL006 at the concentrations of 300 µg/mL and 400 µg/mL. However, with free ZL006 (100 µg/mL to 400 µg/mL), it showed no obvious concentration dependency. At different concentration points, the uptake of SHp-P-LPs/ZL006 and T7&SHp-P-LPs/ZL006 were obviously higher than that of P-LPs/ZL006 and T7-P-LPs/ZL006, and they are 15

ACCEPTED MANUSCRIPT both higher than that of free ZL006. Meanwhile, to study the stroke homing function of SHp, PC-12 cells were incubated with SHp-P-LPs/ZL006 and P-LPS/ZL006

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without glutamate to simulate the uptake in normal central nervous system. It

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indicated that the uptake of both liposomes were concentration-dependent. Furthermore, when cells were incubated without glutamate, the uptake of SHp-P-LPs/ZL006 and P-LPS/ZL006 were at a similar level (Fig. 3C). In other words,

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SHp did not exhibit its homing function to intact central nervous system (CNS).

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Fig.3 The ratio of ZL006 loaded liposomes transported across BBB in vitro (n=6) (A).***P˂ 0.001 compared with P-LPs/ZL006 group or T7-P-LPs/ZL006 group. Cellular uptake 17

ACCEPTED MANUSCRIPT of free ZL006, P-LPs/ZL006, T7-P-LPs/ZL006, SHp-P-LPs/ZL006 and T7&SHp-P-LPs/ZL006 at the concentrations ranged from 100 µg/mL to 400 µg/mL in the glycine incubated PC-12 cells (n=6) (B). **P˂ 0.01, ***P˂ 0.001 compared with P-LPs/ZL006 group or T7-P-LPs/ZL006 group

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at the same concentration. Cellular uptake of P-LPs/ZL006 and SHp-P-LPs/ZL006 in PC-12 cells treated with or without glutamate and glycine (n=6) (C). **P˂ 0.01, ***P˂ 0.001 compared with

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GLU-DSPE group, DSPE group and SHp group at the same concentration. DSPE: P-LPs/ZL006 without glutamate; GLU-DSPE: P-LPs/ZL006 with glutamate; SHp: SHp-P-LPs/ZL006 without glutamate; GLU-SHp: SHp-P-LPs/ZL006 with glutamate.

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The detrimental cascades of events during cerebral ischemia are complex and not

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fully understood, but it is generally accepted that a pathological release of glutamate from neurons plays a central role in mediating subsequent neuronal cell injury and

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death[33]. Previous evidence strongly suggested that cerebral ischemic injury was

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related to massive influx of extracellular Ca2+[34]. Although the mechanism of SHp

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homing in the excitotoxicity of ischemic injury models is not clear, it is likely to be related to physical-chemical properties of the peptides. It showed an exciting result

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that the SHp could exhibit effective homing and penetration abilities when PC-12 cells were stimulated by glutamate (Fig. 3B). However, there were no significant differences in cellular uptake of ZL006 between SHp-modified and plain liposomes were observed when cells were incubated in the absence of glutamate (Fig. 3C). In other words, it was demonstrated that SHp could exhibit a better homing function and penetrate ability in the presence of glutamic acid exposure caused cell excitotoxicity. And it was a good explanation for why the SHp-P-LPs accumulated in ischemic lesion rather than normal cerebral hemisphere. It could be speculated that the changes of microenvironment caused by cerebral ischemic injury, such as pathological release 18

ACCEPTED MANUSCRIPT of glutamate, massive influx of extracellular Ca2+ or oxidative stress, result in good stroke homing targeting function of SHp. Meanwhile, in ischemic stroke, glutamate

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receptors would be over activated and became a critical mechanism for neuronal death.

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It is reported that over activation of N-methyl-D-aspartate (NMDA) type glutamate receptor induced calpain-mediated truncation of metabotropic glutamate receptor mGluR1α, resulting in suppression of its neuroprotective signaling pathway[35].

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Considering SHp could exhibit a better homing function with the existence of extra

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glutamic acid, it was speculated that over activation of glutamate receptors might be the function site of SHp.

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In our study, dual-targeting liposome exhibited the highest efficiency of both

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penetrating BBB and homing to ischemia area. T7 peptide can specifically bind to

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TfR on the surface of BCEC cells and SHp can show specific affinity to glutamate stimulated PC-12 cells.

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3.5. Ex vivo fluorescent image of ischemic brain To validate T7&SHp-P-LPs for targeting to MCAO rat brains, we performed ex vivo fluorescent imaging of the ischemic brains. P-LPs/DiR, T7-P-LPs/DiR, SHp-P-LPs/DiR and T7&SHp-P-LPs/DiR were injected immediately after the operation. Previous research have reported that nanoparticles may across broken BBB induced by ischemia/reperfusion[36, 37]. A small amount of P-LPs and SHp-P-LPs accumulated into brain due to the broken BBB. As shown in Fig. 4, compared with the P-LPs group, the fluorescence intensities of SHp-P-LPs group and T7&SHp-P-LPs group in ischemic region were much higher than other parts of brain owing to the 19

ACCEPTED MANUSCRIPT specific distribution into ischemic regions of SHp. Besides, T7 modified liposomes could deliver more DiR across the BBB into brain. So that T7&SHp-P-LPs group

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showed higher fluorescence intensity than T7-P-LPs group and SHp-P-LPs group.

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The results indicated T7 and SHp-modified dual-targeting liposomes could penetrate

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BBB and target to the ischemic region of brain effectively and consistently.

Fig.4 Ex vivo fluorescent image of DiR-labeled P-LPs, T7-P-LPs, SHp-P-LPs and T7&SHp-P-LPs in the ischemic brain at 6 and 24 h. Arrow: Ischemic cerebral hemisphere (A). Semi-quantitative analysis of the fluorescent intensity of the formulations in 6h (B) and 24h (C) (n=3). ***P<0.001 20

ACCEPTED MANUSCRIPT compared with the other groups. P-LPs: DiR-labeled P-LPs; T7-P-LPs: DiR-labeled T7-P-LPs; SHp-P-LPs: DiR-labeled SHp-P-LPs; T7&SHp-P-LPs: DiR-labeled T7&SHp-P-LPs.

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3.6. In vivo anti-ischemic stroke efficacy

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The infarct volume was measured 24 h after MCAO using TTC-staining and

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neurological deficit scores in MCAO rats. Rats in the sham-operated group had no infarct and neurological deficit score (Fig. 5). By contrast, in the MCAO model group,

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the infarct volume and neurological deficit score were significantly higher. As shown in Fig. 5, treatment with T7&SHp-P-LPs/ZL006, MCAO rats showed decreases in

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infarct volumes and amelioration of neurological deficits compared with those of

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P-LPs/ZL006 group (P<0.01). These results suggested that the ZL006 displayed a

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excellent neuroprotective effect on ischemic stroke. Furthermore, ZL006 loaded T7 and SHp functionalized dual-targeting liposome exhibited greater neuroprotective

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effect than equivalent doses of free ZL006, plain ZL006 liposome and SHp modified

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liposomes on the surgical MCAO injury.

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Fig.5 Effect of SHp-P-LPs on brain infarct volume and neurological deficits at 24 h after MCAO

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in rats. Representative TTC-stained brain sections of Sham-operated group, MCAO group, free ZL006 group, P-LPs/ZL006 group, T7-P-LPs/ZL006 group, SHp-P-LPs/ZL006 group and T7&SHp-P-LPs/ZL006 group were shown in the figure (A). The non-ischemic region is red, and the infarct region appears in white. Quantification of brain infarct volume (B), Neurological scores of rats after cerebral ischemia (C) were shown. Data are expressed with mean ±SD (n=9). *P<0.05, **P<0.01, ***P<0.001 compared with MCAO group. ##P˂ 0.01 compared with P-LPs group. ZL006:

free

ZL006;

P-LPs:

P-LPs/ZL006;

T7-P-LPs:

T7-P-LPs/ZL006;

SHp-P-LPs:

SHp-P-LPs/ZL006; T7&SHp-P-LPs: T7&SHp-P-LPs/ZL006.

3.7. Effect of SHp-P-LPs on neuronal apoptosis TUNEL labeling was almost undetectable in the cerebral hemispheres of the sham group (Fig. 6). Focal cerebral ischemia induced an obvious increase in the 22

ACCEPTED MANUSCRIPT incidence of TUNEL positive cells. The apoptotic cells in MCAO group exhibit significant pyknosis and brown staining (Fig. 6A). Although no significant difference

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was found between the free ZL006 group (43.6±4.6%) and the P-LPs/ZL006 group

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(45.3±4.1%), the both groups exhibited a less rate of apoptosis compared with MCAO group (58.2±5.7%) in Fig. 6B. These results indicated that free ZL006, P-LPs/ZL006, T7-P-LPs/ZL006 and SHp-P-LPs/ZL006 had some extent of protective effect against

other

groups,

T7&SHp-P-LPs/ZL006

(Fig.

6)

significantly

restrained

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the

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neural cells apoptosis induced by ischemia/reperfusion insult. But compared with all

ischemia-induced neural cells apoptosis, as indicated by a remarkable decrease

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TUNEL-positive cells (11.3±1.8%, P<0.001).

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There is a concern about the toxicity of targeting liposomes. Therefore, the

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systemic toxicity of T7-P-LPs, SHp-P-LPs and T7&SHp-P-LPs in mice was investigated in this study. No death and serious body weight loss were found in the

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both of saline and kinds of liposome treated groups during the study period. There was no significant difference of the aspartate transaminase (AST), alanine transaminase (ALT), urea nitrogen (BUN) and creatinine levels between the four groups (Table S2, Supporting Information), indicating that there was no serious inflammatory response caused by liposomes. Besides, major tissues sections of T7-P-LPs, SHp-P-LPs and T7&SHp-P-LPs treated group (including brain, heart, liver, spleen, lung and kidney stained with H&E) showed no apparent changes in cellular structures, necrosis, congestion or hydropic degeneration compared with the saline group (Figure S4, Supporting Information). Taken together, the results exhibited that 23

ACCEPTED MANUSCRIPT multiple dosing of these liposomes had minimal impact in these tissues and did not

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cause systemic toxicity.

Fig.6 Effect of T7&SHp-P-LPs/ZL006 on neuron apoptosis. Representative photomicrographs of sham-operated group, MCAO group, free ZL006 group, P-LPs/ZL006 group, T7-P-LPs/ZL006 group, SHp-P-LPs/ZL006 group and T7&SHp-P-LPs/ZL006 group (A). Scale bar: 20 µm. TUNEL-positive

cells

(brown

staining)

were

counted

under

magnification.

(n=3).

TUNEL-positive cells were obviously observed at 24 h after MCAO, which were significantly decreased by T7&SHp-P-LPs/ZL006 (B). Data are expressed as means±SD. ***P<0.001 compared with P-LPs/ZL006 group. ##P<0.01 compared with MCAO group. ΔΔΔP<0.001 compared with sham-operated group.

4. Conclusion 24

ACCEPTED MANUSCRIPT In conclusion, we conjugated the T7 and SHp to the surface of PEGylated liposomes via a maleimide-thiol reaction and demonstrated that the T7&SHp-P-LPs

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could effectively deliver ZL006 across the BBB and into the ischemic lesion of

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cerebral ischemia/reperfusion injury in vivo. Cellular assay indicated that T7&SHp-P-LPs/ZL006 could obviously penetrate BBB and enhance cellular uptake than that of unmodified P-LPs when cells were endured excitotoxicity by excitatory

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amino acid and reduce cells apoptosis caused by glutamate. The present results

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indicated that ZL006 loaded dual-targeting liposome could significantly ameliorate infarct volume, neurological deficit and histopathological severity in MCAO induced

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cerebral ischemia/reperfusion injury. Furthermore, in ex vivo fluorescent images of

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ischemic brains, T7&SHp-P-LPs/DiR exhibited desirable BBB penetrating and stroke

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homing functions with significantly increased delivery efficacy to ischemic region. Our results demonstrated that T7&SHp modified dual-targeting liposome could

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transport more drugs in to brain and selectively deliver drugs to the ischemic tissue, enhancing the local concentration of the drugs while reducing their side effects. Acknowledgements This work was supported from the National Natural Science Foundation of China (81273457, 81302710), Natural Science Foundation of Jiangsu Province (BK2012843, BK2012445), the ordinary university natural science research project of Jiangsu Province (13KJB350004) and the Excellent Young Teacher Project of Nanjing Medical University (2015RC16). We are acknowledged the sponsorship of Jiangsu Overseas Research & Training Program for University Prominent Young & 25

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Middle-aged Teacher and Presidents.

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Graphical abstract

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