T7 Peptide-Functionalized PEG-PLGA Micelles Loaded with

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T7 peptide-functionalized PEG-PLGA micelles loading with carmustine for targeting therapy of glioma Yunke Bi, Lisha Liu, Yifei Lu, Tao Sun, Chen Shen, Xinli Chen, Qinjun Chen, Sai An, Xi He, Chunhui Ruan, Yinhao Wu, Yujie Zhang, Qin Guo, Zhixing Zheng, Yaohua Liu, Meiqing Lou, Shiguang Zhao, and Chen Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b05572 • Publication Date (Web): 28 Jul 2016 Downloaded from http://pubs.acs.org on July 31, 2016

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ACS Applied Materials & Interfaces

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T7

peptide-functionalized

PEG-PLGA

micelles

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loading

with

carmustine for targeting therapy of glioma

Yunke Bi, 1,3 Lisha Liu, 2 Yifei Lu, 2 Tao Sun, 2 Chen Shen, 1 Xinli Chen, 2 Qinjun Chen, 2

Sai An,

2

Xi He, 2 Chunhui Ruan, 2 Yinhao Wu, 2 Yujie Zhang, 2 Qin Guo, 2 Zhixing

Zheng, 1 Yaohua Liu, 1 Meiqing Lou, 3 Shiguang Zhao *,1, Chen Jiang *,2

1

Department of Neurosurgery, First Affiliated Hospital of Harbin Medical University,

Harbin, Heilongjiang, 150001, PR China 2

Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of

Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, PR China State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, 201203, PR China 3

Department of Neurosurgery, Shanghai First People’s Hospital, Shanghai Jiao Tong

University, School of Medicine, Shanghai, 201620, PR China

Keywords: Receptor-mediated endocytosis, Transferrin receptor, Blood-brain barrier, Micelle, BCNU, and Glioma

ABSTRACT: Glioma is regarded as the deadliest and most common brain tumor because of the extremely difficult surgical excision ascribed from its invasive nature. In addition, the natural of blood-brain barrier (BBB) greatly restricts the therapeutics’ penetrating into the central nervous system. Carmustine (BCNU) is a widely used anti-glioma drugs in clinical applications. However, its serious complications prevent it from being applied in clinic to some extent. Thus, it’s in urgent demand to explore novel BCNU delivery systems specially designed for glioma. The development of polymeric nanoparticles offers a favorable alternation to serve this purpose. Particularly, the use of poly-(lactic-co-glycolic acid) (PLGA) has shown advantageous

ACS Paragon Plus Environment

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for its favorable biodegradability and biocompatibility that ensure safe therapies. In this study, T7 peptide conjugated, BCNU-loaded micelles were constructed successfully via the emulsion-solvent evaporation method. The micelles were characteried by TEM and DLS in detail, and the capacity of BBB-crossing was studied. The in vivo detecting results of targeting effect using the BODIPY probe evidenced that T7-modified micelles showed a more pronounced accumulation, and accumulated in tumor more efficiently than that of unconjugated. Meanwhile, the targeting group exhibited a best curative effect accompany with the gentlest loss in body weight, the smallest tumor size and an obviously prolonged survival time among the groups. In the near future, we believe the targeted delivery system specially designed for BCNU is expected to provide sufficient evidences to end up with clinical trials.

INTRODUCTION Glioblastoma multiforme (GBM), as the most frequent and aggressive subtype of malignant brain tumor, is notorious for its high morbidity and mortality. Despite the combination therapy, patient prognosis is far from satisfactory.1-3 Among all the therapies, systemic chemotherapy plays an indispensable part in the treatment of GBM, but the existence of blood-brain barrier (BBB) poses a major obstacle to delivering active chemotherapeutic agents to the residual site within the central nervous system (CNS).4,5 Besides, additional limitations such as short half-lives, insufficient local drug concentrations and significant hematologic toxicity significantly restrict the clinic application of various systemic agents. Carmustine (BCNU), 6-8 with low molecular weight and good lipid solubility, has been widely used for the treatment of glioma as its favorable permeability in crossing BBB. However, the short elimination half-life and poor selectivity would increase the systemic administration frequency and result serious adverse effects, including hepatic toxicity, myelosuppression and pulmonary fibrosis.9 As a local delivery strategy, BCNU-loaded polyanhydride wafers (Gliadel®) have been applied in clinic



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for several years with the approval of FDA. 10,11 Nevertheless, some complications related with the implantation of BCNU wafers, such as seizures, cerebral edema, cyst formation or infection, are fully recognized.12-14 To some extent, these adverse factors prevent BCNU from being applied in clinic. In recent years, the emergence of nanomedicines and the progress of brain targeting delivery systems bring hope for the treatment of glioma patients.15-18 However, long-term toxicity of the new biomaterials involved is still under discussion,

19,20

leading to a long period ahead for clinical application. Meanwhile, brain targeting drug delivery systems are, unfortunately, a really urgent need for clinic. As an FDA approved biomaterial, Poly (lactide-co-glycolides) (PLGA) can be degraded into non-toxic lactic acid in vivo.21,22 Besides, PLGA, which is regarded with long-term clinical experience, favorable degradation characteristics and sustained release ability, is ranked as one the most popular biodegradable polymers.23 The hydrophilic block of PEG could prolong the systemic circulation time and minimize opsonization which leads to the non-specific uptake in normal tissue.24,25 There are previous studies that concerning PLGA-PEG micelles used for glioma treatments.26-28 Thus, PLGA-PEG was adopted as delivery vehicle in order to realize the clinic use as soon as possible. Polymeric micelles with modest modification at the surface, a promising and efficient delivery system, can carry drugs across the BBB into the glioma site (Scheme.1). 29,30 We intended to construct a micelle drug delivery system loading BCNU for targeting treatment of glioma. As an alternative to conventional chemotherapy, active targeting drug delivery systems has provoked particular interest to facilitate drug delivery to tumor cells.31-33 Modern strategies have been explored, which highlights the covalent attachment of the targeting ligands to the nanoparticle surface with the aim of transporting the payload drug across the BBB via adsorptive or receptor-mediated transport routes. 34,35

Previous reports have indicated that the expression of transferrin (Tf) receptors on

the brain capillaries endothelial cells (BCECs) and some malignant tumor cells is observably higher than other normal cells.36 Based on the natural, Tf-mediated drug


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delivery system has been developed over years. However, the endogenous Tf was also high concentrations, which may bring a competitive inhibition effect.37 Fortunately, T7 peptide (sequence His-Ala-Ile-Tyr-Pro-Arg-His) has a similar targeting ability without competing with endogenous Tf for the receptor binding. Interestingly, the binding of Tf with receptors will promote the transportation of T7 peptide. 38-41 Thus, T7 peptide can be regarded as an ideal exogenous targeting ligands that specifically binds to Tf receptor with a dual-targeting function. In order to accelerate the clinical translation of nanomedcine, PLGA–PEG, as a biodegradable and biocompatible amphipathic copolymer, was chose to prepare BCNU-loaded micelles in this study. BCNU loaded PLGA-PEG micelles were constructed through an emulsion-solvent evaporation method. PLGA served as the core of micelles carrying BCNU, with T7 covalently modified on the surface. The physicochemical properties of micelles, including particle size, surface morphology and drug loading were measured by TEM, HPLC and Malvern Particle Sizer. BCECs and U87 were selected as the cell models for the experiments in vitro. The cytotoxicity, cell cycle, cellular uptake and related mechanism with the treatment of micelles were assessed. Furthermore, the orthotopic glioma model was established on nude mice to evaluate the therapeutic efficacy and safety by the investigation of weight change, tumor size and median survival time. Besides, H&E staining and immunohistochemistry were tested to further evaluate the therapy effect at a molecular level and study the safety, toxicity and side effects of micelles on nude mice. We believe this study can pave an avenue to targeted therapy of glioma.

EXPERIMENTAL SECTION Materials BCNU was commercial available from Meilune Biological co., Ltd (Dalian, China). T7 peptide, with a cysteine on N-terminal, was purchased from Chinese Peptide Company (Hangzhou, China). Poly (D, L-lactic-co-glycolic acid) (PLGA-COOH, Mw 4000) was purchased from PURAC (Holland). Α-Malemidyl-polyethyleneglycol-ω



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-amine (Mal-PEG5000-NH2) was purchased from JenKem Co., Ltd (Beijing, China). MTT,

Dimethyl

Formamide

(DMF),

1-Ethyl-3-

(3-dimethylaminopropyl)-

carbodiimide(EDC), N-hydroxysuccinimide (NHS), filipin, colchicines, phenylarsine oxide, Hoechst 33342 and coumarin-6 were all purchased from Sigma-Aldrich (St. Louis, USA). BODIPY was synthesized as previously described. Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco BRL (Carlsbad, CA, USA). Non-essential amino acid,

trypsin-EDTA

(0.25%), streptomycin and penicillin were obtained from Invitrogen Co., USA. Centrifugal filters were purchased from Amicon Ultra (Millipore, MA, USA). Rabbit cleaved caspase-3 antibody and β-actin antibody were obtained from Cell Signaling Technology (Beverly, USA). The other chemicals unmentioned were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Cell lines and animals The BCECs were a gift from Prof. J. N. Lou (the China-Japan Friendship Hospital), which were expanded and maintained in special DMEM supplementary with a heat-inactivated FBS (20%), epidermal cell growth factor (100 μg/mL), glutamine (2 mmol/L), heparin (20 μg/mL), insulin (40 μU/mL), streptomycin (100 μg/mL), penicillin (100 U/mL), and cultured under a humidified environment incubator (Thermo HERA cell, USA) at 37 °C with an environment of 5% CO2. Human glioma cell lines U87 MG were a gift from Prof. L. Y. Feng (Chinese Academy of Sciences). Glioma U87 cells expressing photinus pyralis luciferase (U87-Luci) were gifts from Doc. N. Zhang (Caliper Life Sciences, A Perkin Elmer Company). Both cell lines were cultured in the DMEM medium, supplementary with streptomycin (100 μg/mL), penicillin (100 U/mL) and fetal bovine serum (FBS, 10%) in a humidified 5% CO2 incubator at 37 °C. BALB/c nude mice (Male, 20 ± 2 g weight preferred) were obtained from Slac Lab Animal Co., Ltd (Shanghai, China) and raised on standard housing conditions by Department of Experimental Animals, Fudan University (Shanghai, China). All experiments of animals were supervised and implemented according to the guidelines


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approved by Fudan University’ ethics committee. Synthesis of T7-PEG-PLGA Maleimide-functionalized copolymer Mal-PEG-PLGA was prepared by linking Mal-PEG-NH2 to PLGA-COOH through a carbodiimide-mediated coupling reaction according to a previous report.42 Generally, excess NHS (23 mg, 0.2 mmol) was added to DMF (7 mL) dissolved with PLGA-COOH (400 mg, 0.1 mmol) to form PLGA-NHS in the presence of EDC (57.3 mg, 0.3 mmol). After that, the mixture was further stirred for 24 h, and the resulted PLGA-NHS was reacted with Mal-PEG-NH2 (500 mg, 0.1 mmol) for 2 h followed by dialysis with a membrane (Mw 5000) for 24 h and lyophilization. Then Mal-PEG-PLGA was conjugated with Cys-T7 (0.1 mg, 0.1 mmol) in DMF (1 mL) for 24 h at room temperature in dark. The Mal groups of PEG-PLGA were then reacted with the thiol groups of Cys-T7. At last, the obtained conjugation T7-PEG-PLGA was carefully purified through a dialysis using a membrane (Mw 5000) and then lyophilized. Preparation of the micelles The BCNU-loaded micelles were obtained via the classic emulsion-solvent evaporation method. Briefly, certain amount of BCNU together with PEG–PLGA or T7-PEG–PLGA (1/10, w/w) dissolved in 2 mL dichloromethane. Then the mixture was stirred for 15 min to allow a slow removal of the organic solvent under vacuum. Then, the resulting T7–PEG–PLGA/BCNU and PEG–PLGA/BCNU micelles were dissolved in 1 mL ddH2O and stirred for 2 h to promote the dissolution. Thereafter, the obtained micelles were filtered using a 0.45 μm cellulose acetate filter. The used micelles need to be freshly prepared in the following experiments. Characterization of micelles The micelles’ characteristics were characterized through nuclear magnetic resonance (NMR) spectroscopy in detail. Generally, T7–PEG–PLGA and PEG–PLGA were lyophilized, re-solubilized by D2O and then analyzed on a 600 MHz NMR spectrometer (Varian, USA). Zeta-potential and mean hydrated diameter of the obtained micelles were measured using the NicompTM 380 ZLS (PSS Nicomp



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Particle Size System, USA). The micromorphology of the micelles was characterized by transmission electron microscope (JEOL JEM-1200EX, Tokyo, Japan). Drug encapsulation efficiency and loading efficiency HPLC was employed to analyze the loading capacity (LC%) and encapsulation efficiency (EE%) for BCNU in micelles. The ultra violet scanning results suggested an absorption peak at 235 nm for BCNU. The content assay of drugs was analyzed on an HPLC system equipped with a C18 column (5 mm, 200 mm, Dikma) and a UV-detector (LC 2010C type, Shimadzu). The adopted mobile phase is mixed methanol/sodium dihydrogen phosphate buffer (0.05 M, pH 7.0)/trifluoroacetic acid (TFA) (55/45/0.2, v/v/v) with 1.0 mL/min flow rate. Under these chromatography conditions, the BCNU’s retention time was found to be 7.0 min. The standard curves of BCNU were created by the determined peak areas vs. known drug concentrations. Then the contents of BCNU encapsulated in micelles were analyzed as follows. The peak area of drugs was measured by HPLC after the supernatant being diluted with methanol, and the concentration was obtained through the respective calibration curves. The drug LC% of micelles was also calculated as previously described.43 In vitro drug release The BCNU’s release kinetics in vitro was studied using a model of Coumarin-6 (Cou-6), considering BCNU is extremely easy to decompose in solution. PBS (pH 7.40) was used as the media. Generally, 100 μL of BCNU loaded micelles were added into 400 μL PBS in 96-well plates and incubated with 100 rpm shaking rate for 48 h. At different time intervals (0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48 h), the samples were analyzed by enzyme standard instrument. All the above operations were repeated in triplicate. Cytotoxicity and cell cycle analyses MTT assay, as a common tool, was used to investigate the cytotoxicity of BCNU loaded micelles to U87 cells. Generally, U87 cells were uniformly seeded in 96-well plates with the density of 5×103 cells per well in the DMEM medium containing FBS (10%). When an 80% confluence was achieved, the cultured cells were treated with


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free BCNU and different micelles solutions at various concentrations (from 0.16 μg/mL to 20 μg/mL). Then the cells were incubated for 24 h at 37 °C, and the medium was removed. After that, the MTT stock solution (20 μl, 5 mg/mL) was added to each group continuing incubation for another 4 h. At last, the supernatants were totally replaced by 120 μl dimethyl sulfoxide (DMSO). Absorbance was measured at 570 nm using a microplate reader (Model ER-8000, Japan). In parallel, 8 wells were treated in each concentration and the averaged absorbance value were normalized to the control cell group (without treatment) and plotted using Origin 8.0. Flow-cytometric analyses were performed to detect cell cycle. In short, U87 cells, seeding in 6-well plate after treatment with free BCNU and different micelles for 24 h, were carefully washed by Hank’s solution, trypsinized and suspended into single-cell mixtures. Then, RNase A (20 μg/mL) was used to treat U87 cells, and Propidium Iodide (PI, 25 μg/mL) was adopted to stained the cells for 1 h before the subjection to the cell cycle analyses. The DNA content was then measured by a flow-cytometric analyses using a FACS Calibur flow cytometer (FACS420). For per experiment, a total of 10,000 events were collected, and the MODFIT software program was employed to analyze the data. Western Blot RIPA buffer (Thermo Scientific, USA) containing PMSF (1%) was used to lyse U87 cells and the protein were extracted. The samples’ concentration were determined by the BCA kit (Thermo Scientific, USA) according to the instructions. 12% SDS-PAGE gels were used to separate the lysates, and PVDF membranes were adopted to transfer protein. Briefly, the membranes were blocked with skim milk (5%) containing Tween-20 (0.1%) for 1 h at r.t., and incubated overnight at 4 °C with rabbit caspase-3 antibody (1/1000, Cell Signaling Technology, USA) and mouse β-actin antibody (1/3000; Santa Cruz, USA). On the next day, the obtained membranes were washed with PBST and further incubated with secondary antibodies (1/5000, Invitrogen, USA) at shaking state for 1 h. At last, the bands were captured through BeyoECL Plus (Beyotime, China) and detected with a BioImaging System. β-actin was used as the



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internal control. Cellular uptake of different micelles and that with different inhibitors The fluorescent inverted microscope (DMI4000 B, LEICA, Germany) was used to evaluate cellular uptake efficiency of different micelles. Considering the unstable characteristic of BCNU, Coumarin-6 (Cou6) was used as the model to study the drug encapsulating ability into the unmodified or T7-modified micelles using the same method as BCNU loaded micelles. BCECs and U87 cells were uniformly seeded in 24-well plates at a density of 4 × 104 cells/well and incubated until an 80% confluence was achieved. Then, both cells were incubated with T7-PEG-PLGA/Cou6 or PEG-PLGA/Cou6 at the 0.5 mg/well concentration measured by PEG-PLGA for 30 min. As for inhibitors groups, different inhibitors including colchicines (2.5 μM), phenylarsine oxide (2.5 μM), filipin (0.5 μg/mL), and 100 × T7 were pre-cultured with the cells for 10 min, following the addition of different micelles treated with a similar condition mentioned above. After that, the supernatants were removed and washed by Hank’s. All the images were captured with the same imaging parameters in order to give a real comparison on the intensity of fluorescence of different formulations. For the quantitative analysis, cells were trypsinized and centrifuged at 1000 rpm for 5 min, then washed by Hank’s solution, re-suspended in PBS and analyzed by a flow cytometer finally (FACS Aria, BD, USA). Intracellular tracking of T7-PEG-PLGA/Cou6 micelles BCECs and U87 cells were uniformly seeded in dishes at a density of 2 × 104 cells for confocal imaging. After 24 h, the cells were incubated with T7-PEG-PLGA/Cou6 and PEG-PLGA/Cou6 micelles (1mg PEG-PLGA, 1 mL/dish) for 15 min, 30 min and 60 min, respectively. Then, Hoechst 33342 (10 μl, 1 mM) was added, 10 min before the preassigned time point. After the incubation, the cells in the well were carefully washed twice with Hank’s and then detected using confocal laser scanning microscopy (Leica TCS SP2, Germany). Cou6 and Hoechst 33342 fluorescence were excited with the 466 and 350 nm wavelength, and the emission was measured in a


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range of 504 nm (Cou6) and 461 nm (Hoechst 33342). Animal model establishment BALB/c nude mice (Male, 22 ± 3 g weight preferred) were obtained from SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and raised under the SPF conditions. To establish the glioma model, nude mice were anaesthetized by Chloral hydrate (10%) and slowly injected with U87-Luci cells (1×105 cells/5 μl ) into the right corpus striatum (1.8 mm lateral, 0.6 mm anterior to the bregma and 3.0 mm in depth) by a stereotactic fixation device using a mouse adaptor. Two weeks later, nude mice were imaged in vivo real-time bioluminescence imaging system (IVIS, Cailper PerkinElemer, USA) for confirmation of the establishment of the glioma model. All animal experiments conducted in the current study were evaluated supervised, and approved by the Ethics Committee. In vivo real-time imaging and micelles distribution For visualizing the in vivo real-time distribution, the fluorescent probe BODIPY, as the NIR dye and another model, was encapsulated into the unmodified or T7-modified micelles using the same method as BCNU micelles. Nine glioma-bearing nude mice were divided into three groups randomly and administrated with saline (200 μL), PEG-PLGA/BODIPY and T7-PEG-PLGA/BODIPY (1 mg/kg) through the tail vein, respectively. The images were acquired by IVIS Spectrum after 24 h of the administration. Then the nude mice were sacrificed, the glioma-bearing brains and major organs were carefully harvested for fluorescent imaging. After imaging, the U87-Luci bearing brains were fixed in 4% paraformaldehyde for 48 h, dehydrated with sucrose solution at different concentrations (15% and 30%) for 24 h sequentially. Thereafter, the brains were embedded in OCT and frozen at -20 °C, sectioned at 14 mm. At last, the slides were stained with DAPI and observed using the confocal microscopy. Anti-glioma efficacy BALB/c nude mice bearing with intracranial U87-Luci glioma were randomly divided into 4 Groups (n = 8) according the fluorescence image obtained by the IVIS



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Spectrum, treating with Saline, free BCNU, PEG-PLGA/BCNU and T7-PEG-PLGA /BCNU (at an equal dose of 1 mg/kg BCNU), respectively, every two weeks for three times. The efficacy of the micelles was further evaluated by imaging the size of glioma, and measuring the body weight and the survival term of the animals after the treatments. TUNEL assay was employed to observe the cell apoptosis in the tumor tissue. Upon the treatment with different BCNU-loaded micelles, the mice were then sacrificed and the obtained brains were carefully sectioned, subjected to TUNEL to observe the broken nuclear DNA fragments. The tissues were stained by DAPI, and then examined using fluorescent microscope (Olympus, Japan). H&E staining and immunohistochemistry analysis Mice were sacrificed after treatment with different BCNU-loading micelles. The major organs were immediately fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 5-mm-thick sections which were stained with H&E for the histological examination. Caspase-3 and Ki67 protein were detected of brain tumor sections by immunohistochemical. Statistical analysis We present all the data as mean ± SD deviation. One-way ANOVA with Bonferroni tests for a multiple-group analysis and unpaired student’s test was used for between two-group comparison and. Statistical significance was defined as p < 0.05.

RESULTS AND DISCUSSION Preparation and characterization of micelles PLGA–PEG was synthesized successfully by linking PLGA-COOH to the amino of NH2–PEG by an EDC/NHS mediated chemical reaction. Peptide T7 was conjugated to PLGA–PEG-MAL to construct the targeting copolymer, T7–PEG–PLGA (Figure S1A). 1H NMR spectrum was adopted to determine the structure and purity. Being different from the 1H NMR spectrum of Mal–PEG–PLGA, the MAL peak (7.0 ppm) disappeared in T7–PEG–PLGA (Figure S1B in the Supporting Information),


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indicating that the complete consumption of the thiol group of T7 peptide by the MAL group. The above results suggested the successful synthesis of T7–PEG–PLGA . BCNU-loaded micelles were constructed by a classic emulsion-solvent evaporation method. The loading parameters and physicochemical properties of different BCNU-loaded micelles were detected (Table S1). The mean size of PEG– PLGA/BCNU and T7–PEG–PLGA/BCNU were 76.85 nm and 83.31 nm (Figure 1A.a, b), respectively, with a narrow distribution (PDI. 0.089 and 0.146). The conjugation with NHS-PEG-MAL or T7 peptide slightly increased the micelles size. The entrapment efficiency (EE%) of BCNU-loaded micelles was around 66.28% and the drug loading (DL) rate was 5.49% without any significant differences between them, indicating that the encapsulation ability was not influenced by the introduction of T7 peptide. The formation was further confirmed by TEM observation. Figure 1A.c, d showed that the micelles were well-dispersed in the aqueous solution and exhibited an obvious core-shell structure. Coumarin-6 (Cou6) is a classic model in studying drug-releasing characteristics. Considering the poor stability of BCNU in solution, we selected Cou-6 as an alternative to detect the drug release from the vehicles in vitro. The release kinetics of formulations was carefully evaluated in PBS (pH 7.4, Figure 1B). A slight burst release was found in the initial 10 h (63% and 78%, respectively). Then the signals of PEG–PLGA/Cou6 group decreased in later time, while the T7–PEG–PLGA/Cou6 group continue releasing until an 80% level, showing a sustained release behavior. This could be ascribed to the increased interactions between the molecules of Cou6 and the targeting peptide in the releasing procedure. Cellular uptake of micelles and related mechanism The cellular uptake of micelles was studied using BCECs and U87 cells. Coumarin-6 (Cou6) was used as the classic fluorescent model. Images under fluorescent microscope showed that both cell lines treated with T7–PEG–PLGA/Cou-6 (10 wt %, 20 wt % and 40 wt % T7 modified) exhibited a higher level of internalization compared with PEG–PLGA/Cou-6 (Figure 2), indicating the dual targeting effect of



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T7 peptide. Consistent with the qualitative analysis, quantitative analysis demonstrated the same concentration-dependent cellular internalization manner (Figure S2B). The mechanism of T7 dependent delivery was investigated through the following experiments both on BCECs and U87 cells (Figure S2A). On one hand, a significant decrease of fluorescence intensity was found in the group pre-treated with free T7 peptide (100 ×) compared with the untreated one. On the other hand, different inhibitors were adopted to further clarify the endocytosis process. The treatment with filipin resulted in the most extent decrease of fluorescent, and the fluorescent intensity declined to some extent by phenylarsine oxide and colchicine inhibition. Consistent with the qualitative analysis, the quantitative analysis demonstrated similar results (Figure S2B). These results demonstrated that T7-modified micelles were uptaken mainly through the caveolae-mediated endocytosis, partly micropinocytosis and clathrinmediated endocytosis. Intracellular tracking of T7–PEG–PLGA/Cou6 micelles BCECs and U87 cells were incubated with micelles over different time points, following the addition of Hoechst 33342. As shown in Figure 3, the blue fluorescent dots represented the nucleus dyed by Hoechst 33342 while the green ones represented T7-PEG-PLGA/Cou6 micelles enter into both cell lines. The results showed that micelles can accumulate in glioma cells in a time-dependent mode, suggesting that the T7-modified micelles can be internalized by BCECs and glioma cells. In vitro cell viability and cell cycle analysis The cytotoxicity of BCNU-loaded micelles to U87 cells was assessed by MTT assay. Figure 4 shows the viabilities of U87 cells after incubation with different micelles for 24 h. It is believed that the IC50 value can reflect the cytotoxicity of different micelles in an indirect way. It was found that the IC50 values of PEG–PLGA/BCNU and T7– PEG–PLGA/BCNU were significantly lower than those of free BCNU, indicating that the encapsulation of BCNU can enhance the cytotoxicity of drug. Furthermore, there is an obvious difference between the IC50 values of PEG–PLGA/BCNU and T7–


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PEG–PLGA/BCNU. The enhancement effect could be attributed to the targeting binding of T7 to U87 cells and sustained release of BCNU. In order to check whether cell death induced by different micelles was related with apoptosis, we detected the exression of cleaved caspase-3 protein by western blot. In Figure S3A, cleaved caspase-3 was accumulated upon treatment, especially in the T7–PEG–PLGA/BCNU group. The effect of BCNU and micelles on the cell cycle and apoptosis of U87 cells was further evaluated by the flow cytometry analysis. Figure S3B in the Supporting Information showed the cell cycle distribution, mainly expressed as a decrease of the cells in G2/M and S phase an accumulation in G0/G1 phase, particularly at the T7– PEG–PLGA/BCNU group, in which the strongest apoptosis was induced. Tissue distribution in tumor-bearing mice Near infrared fluorescence (NIR) optical imaging technology was utilized to monitor the tumor targeting efficiency of BODIPY loaded micelles in U87-Luci bearing mice in vivo 2D (Figure 5A) and 3D distribution (Figure 5B). At the 21st day after implantation, the mice were administrated with T7–PEG–PLGA/BODIPY and PEG– PLGA/BODIPY via the tail vein. In vivo images were captured 24 h after the injection (Figure 5A. a). Compared to PEG–PLGA/BODIPY treated mice, an obvious NIR signal accumulation was found at the mice’s tumor region treated with T7–PEG– PLGA/BODIPY, which was mainly due to the specific and strong binding of T7 to BBB and brain tumors. After that, glioma bearing brains and major organs were excised for ex vivo imaging to reveal the tissue distribution (Figure 5A. b, c and Figure S5A). A clearer result was found in the ex brain imaging (Figure 5A. b, c). Besides, the amounts of T7–PEG–PLGA/BODIPY were less in liver compared to PEG–PLGA/BODIPY group, suggesting a lower toxicity to liver. While the concentrations of micelles in heart, spleen, lung and kidney was similar in both groups. Confocal images of the tumor tissue also revealed that the tumor accumulation of T7–PEG–PLGA/BODIPY was more than that of PEG– PLGA/BODIPY group (Figure S4B).



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In situ tumor apoptosis detection TUNEL assay was used to detect the apoptosis of glioma cells induced by different micelles. As illustrated in Figure 6, obvious apoptosis phenomenon was found in the groups treated by saline, while a few apoptosis was detected in the group treated with free BCNU. The group treated with PEG–PLGA/BCNU arouses a relatively obvious apoptosis phenomenon. An obvious apoptosis phenomenon was noticed inside the tumor site in the T7–PEG–PLGA/BCNU treated group. Antitumor efficacy We used glioma model to evaluate the efficacy of targeting therapy in vivo (Figure 7A). Luciferase expression in the glioma cells was visualized with the injection of luciferin and imaged by IVIS. The luminescence intensity indicated the growth of the U87-Luci glioma. A decrease in luminescence intensity was detected in the mice treated with free BCNU and different micelles at the day 7 after injection. However, we found luminescence intensity was increased in BCNU and PEG-PLGA/BCNU group while the T7-PEG-PLGA/BCNU group was decreased, on the 17th day after 3 times administration. These data indicate that T7-PEG-PLGA/ BCNU micelles can induce significant inhibition on the growth of glioma in vivo. The clinically therapeutic benefits of cancer patients are mainly determined by life quality and prolonged survival time. In order to evaluate the antitumor efficacy in animal models, body weight and overall survival of the mice were evaluated (Figure 7B, C). The only saline-treated group resulted a rapid loss in body weight and a relatively early death. Compared with the control group, both free BCNU group and PEG-PLGA/BCNU group shown a prolonged survival time, while the loss of body weight was gentler. As expected, the T7-PEG-PLGA/BCNU group exhibited the longest survival time, which may be attributed to the targeting inhibition of tumors and reduced toxicity to normal organs. H&E staining and immunohistochemistry analysis H&E staining and immunohistochemistry were tested to further evaluate the therapy effect of different micelles. Figure S5A in the Supporting Information showed the


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brain tumor pathology, from which we can see the boundary of tumor and normal tissues.

On

histological

sections,

we

found

that

tumors

treated

with

T7-PEG-PLGA/BCNU displayed the highest apoptosis rate of tumor cells as reflected by accumulated caspase-3 (Figure 8). In addition, proliferation was remarkably reduced in tumors treated with T7-PEG-PLGA/BCNU as reflected by a reduced staining for Ki-67 (Figure 8). With the aim of the safety study, toxicity and side effects of different micelles to nude mice, we carried out a histology observation on heart, liver, spleen, lung and kidney by H&E staining. Histological analysis indicated that no significant toxic pathological changes were detected (Figure S5B). CONCLUSION In summary, we established a BBB crossing and glioma tissue penetrating system with T7 as the targeting ligand for improved anti-glioma efficacy. Both the in vitro and in vivo experiments verified that T7-PEG-PLGA/BCNU acted more efficiently than PEG-PLGA/BCNU and free BCNU. Due to the outstanding BBB-targeting and glioma-penetrating

efficiency,

T7

peptide

modified

micelles,

which

bind

specifically to the Tf receptors overexpressed in BBB and glioma cells, mainly concentrate on the glioma with little in major organs. In other words, the toxicity and side effects of T7-modified micelles was the least among the group, indicating this drug delivery system was relatively ideal for the treatment of glioma. In short, accumulating evidence confirmed that this glioma targeting system might facilitate anti-glioma drug delivery and promote nanomedicines accumulation and penetration into tumor site, which would be of pronounced significance for the therapy of glioma. In the future study, we will keep on working on the structure-property-relationship and know-how knowledge based on the tumor’s metabolism and microenvironment with the hope of clinical applications.



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ASSOCIATED CONTENT Supporting Information Characterizations of micelles; synthetic steps and 1H NMR spectrum of micelles; exploration of cellular uptake mechanism; cell viability and cell cycle analysis; in vivo and ex vivo distribution of BODIPY loaded micelles; H&E staining of U87-bearing mice brain and major organs.

AUTHOR INFORMATION Corresponding Author *1Email: [email protected]. Tel/Fax: +86-0451-85552799 *2Email: [email protected]. Tel/Fax: +86-21-5198-0079 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the grant from National Basic Research Program of China (973 Program, 2013CB932500), National Science Fund for Distinguished Young Scholars (81425023), National Natural Science Foundation of China (NO. 81172993 to Chen Jiang, NO. 81472368 to Shiguang Zhao, NO. 81372701 to Yaohua Liu), National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (NO. 2013BAH06F04 to Shiguang Zhao), Shanghai Sailing Programe (NO. 16YF1400900 to Tao Sun) and Scientific Research Foundation of Fudan University for Talent Introduction (NO. JJF301103 to Tao Sun).

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Scheme 1. Receptor mediated transcytosis of BCECs and receptor mediated endocytosis of U87 cells for T7-PEG-PLGA/BCNU micelles. (A) Receptor mediated transcytosis of BCECs. (B) Receptor mediated transcytosis of glioma cells.


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Figure 1. The physicochemical properties of BCNU-loaded micelles. (A) The particle size distribution of BCNU-loaded micelles was analyzed by (a,b) dynamic light scattering and (c,d) transmission electron microscope. (B) In vitro drug release properties of Coumarin 6 from micelles in PBS at pH 7.4. Data are mean ±SE (n=3).



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Figure 2. Cellular uptake of Cou 6 loaded micelles and related mechanism exploration. Cellular uptake of (a) PEG-PLGA/Cou 6, (b) 10% T7-PEG-PLGA/Cou 6, (c) 20% T7-PEG-PLGA/Cou 6 and (d) 40% T7-PEG-PLGA/Cou 6 on BCECs and U87 cells were examined by fluorescence microscope after 30 min incubation. Green: Cou 6 loaded micelles. Original magnification: ×100.


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Figure 3. Intracellular tracking of T7–PEG–PLGA/Cou6 micelles in (A) BCECs and (B) U87 cells. Images were taken after incubated with micelles for 15min, 0.5h and 1h, and observed by confocal microscopy. Green spots represent Cou6 loaded micelles, while blue spots are cell nucleus.



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Figure 4. In vitro cell viability analysis. Viability of U87 cells treated with free BCNU, PEG-PLGA/BCNU and T7-PEG-PLGA/BCNU at different concentrations for 24h. The IC50 of the T7-PEG-PLGA/BCNU was 3.902 mg/mL, which was lower than that of PEG-PLGA/BCNU (7.351 mg/mL) and free BCNU (15.22 mg/mL).


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Figure 5. In vivo and ex vivo distribution of BODIPY loaded micelles after intravenous administration. Images were taken at 24 h after injection. (A) (a) In vivo imaging of U87-bearing nude

mice

administrated

with

saline

(left),

PEG-PLGA/BODIPY

(middle)

and

T7-PEG-PLGA/BODIPY (right). Images were taken 24 h after administration. (b) Ex vivo photos of U87-bearing brain. (c) Ex vivo brain fluorescence imaging of BODIPY loaded micelles. The red arrow indicate non-targeting micelles could penetrate into the tumor site partly due to the EPR effect.

(B) 3D real-time fluorescence images of glioma model nude mouse injected with

BODIPY loaded micelles at 24 h.



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Figure 6. TUNEL assay of frozen sections of brain tumors after treatment with different micelles. Green: TUNEL-stained apoptosis cells. Blue: DAPI-labelled nucleus. Dashed line: boundary between normal brain section and glioma section. N: normal brain section; G: glioma section. Original magnification: 100×.


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Figure 7. Anti-glioma efficacy of different micelles on model mice. (A) Real-time bioluminescence images of glioma model nude mice injected with saline, free BCNU, PEG-PLGA/BCNU and T7- PEG-PLGA/BCNU micelles on Day 0, Day 7 and Day17 respectively. (B) Body weight change of model mice. (C) Kaplane Meier survival curves of mice bearing U87 MG tumors.



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Figure 8. Immunohistochemistry analysis for caspase-3 and Ki-67 proteins of brain tumor in nude mice injected with saline, free BCNU, PEG-PLGA/BCNU and T7-PEG-PLGA/BCNU.


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Table of Contents Graphic


T7 Peptide-Functionalized PEG-PLGA Micelles Loaded with

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