A Bacteria Deriving Peptide Modified Dendrigraft Poly-l-lysines (DGL

Jun 25, 2014 - Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Ministry of Health, Beijing, People's Republic of China. ABSTR...
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A Bacteria Deriving Peptide Modified Dendrigraft Poly‑L‑lysines (DGL) Self-Assembling Nanoplatform for Targeted Gene Delivery Yang Liu,†,‡ Xi He,†,‡ Yuyang Kuang,† Sai An,† Chenyu Wang,† Yubo Guo,† Haojun Ma,† Jinning Lou,§ and Chen Jiang*,† †

Key Laboratory of Smart Drug Delivery, Ministry of Education, School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203, People’s Republic of China § Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Ministry of Health, Beijing, People’s Republic of China ABSTRACT: Achieving effective gene therapy for glioma depends on gene delivery systems. The gene delivery system should be able to cross the blood−brain barrier (BBB) and further target glioma at its early stage. Active brain tumor targeted delivery can be achieved using the “Trojan horse” technology, which involves either endogenous ligands or extraneous substances that can recognize and bind to specific receptors in target sites. This method facilitates receptormediated endocytosis to cross the BBB and enter into glioma cells. Dendrigraft poly-L-lysines (DGLs), which are novel nonviral gene vectors, are conjugated to a peptide (sequence: EPRNEEK) derived from Streptococcus pneumonia, a pathogen causing meningitis. This process yields peptide-modified nanoparticles (NPs) after DNA loading. Cellular uptake and in vivo imaging results indicate that EPRNEEK peptide-modified NPs have a better brain tumor targeted effect compared with a pentapeptide derived from endogenous laminin after intravenous injection. The mechanism of this effect is further explored in the present study. Besides, EPRNEEK peptide-modified NPs also exhibited a prolonged median survival time. In conclusion, the EPRNEEK peptide-modified DGL NPs exhibit potential as a nonviral platform for efficient, noninvasive, and safe brain glioma dual-targeted gene delivery. KEYWORDS: brain-targeted, tumor-targeted, gene delivery, laminin receptor, nanoparticles

1. INTRODUCTION Glioma is one of the most common tumors in the central nervous system (CNS). Glioma is graded from I (benign) to IV (highly malignant), according to the severity of the disease.1 The pathological conditions of glial tumors vary in different stages. Thus, different strategies should be applied in designing a drug delivery system for targeting glioma. In the treatment of low-grade glioma, the drug delivery system should be able to cross the blood−brain barrier (BBB) and further target glial cancer cells.2 However, drugs especially for macromolecular proteins and genes are excluded from the brain when administered intravenously because of the BBB, which is formed mainly by brain capillary endothelial cells (BCECs).3 Much research has been done to achieve a desirable delivery of drugs to glioma by using active-targeting ligands, such as transferrin,4 dehydroascorbic acid (DHA),5 and angiopep-2.6 In addition to these endogenous substances, certain pathogens can cross the BBB in a transcellular or paracellular manner and cause CNS infection.7 Various infectious agents, including prions and certain neurotropic viruses, bind to the laminin receptor, thereby determining tropism for the CNS.8 The expression of laminin receptors is increased in most adult neurons as well as glial cells.9 Laminin receptors also have an important function in tumor invasion and metastasis.10 The © 2014 American Chemical Society

above evidence indicates that laminin receptors can further facilitate the laminin receptor ligands or their modified drug delivery system accumulating in the glioma after crossing the BBB. Laminin receptors are found to initiate bacterial contact with the BBB in experimental meningitis models.11 The critical sequence enabling the pneumococcal CbpA domain to bind to the laminin receptor is the sequence EPRNEEK. Considering the important function of binding to the laminin receptor in initiating intimate contact between the circulating bacterial meningeal pathogens and the BBB cells,9,12 we hypothesize that the short peptide (sequence: EPRNEEK) favors laminin receptor binding in the BBB. This process further results in preferable BBB translocation and glioma-targeted accumulation. Viral gene vectors have been proven efficient; however, safety concerns have somehow prevented further applications.13 Special Issue: Recent Molecular Pharmaceutical Development in China Received: Revised: Accepted: Published: 3330

January 28, 2014 May 4, 2014 June 25, 2014 June 25, 2014 dx.doi.org/10.1021/mp500084s | Mol. Pharmaceutics 2014, 11, 3330−3341

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Figure 1. Basic synthetic route of DGLs-PEG-peptide/DNA NPs. (A) Synthetic route of DGLs-PEG-peptide vehicles. (B) Preparation of DGLsPEG-peptide/DNA NPs.

Dendrigraft poly-L-lysines (DGLs), a new kind of synthetic polymers consisting of lysine,16,17 have been employed as a gene vector because of their degradability and rich external amino groups that can encapsulate plasmid DNA through electric interactions. They can also be modified with polyethylene glycol (PEG) and targeting ligands, thereby rendering

Synthetic polymers used in nonviral approaches have been investigated intensively. Polyamidoamine (PAMAM)14 and poly(ether imide) (PEI)15 have shown their potential in gene delivery because of their high encapsulation efficiency. However, the high cytotoxicity drawn by their poor degradability has enormously confined their application. 3331

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Clinical Medicine Research Institute of the Chinese-Japanese Friendship Hospital). Primary BCECs were cultured as described previously.20 Cells used in this study were between passage 10 and passage 20. The U-87 MG human glioblastomaastrocytoma cell line (ATCC number: HTB-14) was kindly provided by Prof. W. Y. Lu (School of Pharmacy, Fudan University). All cells were cultured at 37 °C under a humidified atmosphere containing 5% CO2. Male Balb/c mice (4−5 weeks old) of 20−25 g body weight and nude mice of 20−25 g body weight were purchased from the Department of Experimental Animals, Fudan University, and maintained under standard housing conditions. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Fudan University. 2.3. Synthesis of the Targeted DGLs-PEG-peptide/ DNA NPs. The basic synthetic route for DGLs-PEG-peptide/ DNA NPs is described in Figure 1. DGLs were reacted with NHS-PEG3500-MAL 1:5 (mol/mol) in PBS (pH 8.0) for 2 h at room temperature. The primary amino groups on the surface of DGLs were specifically reacted with the NHS groups of the bifunctional PEG derivative. The resulting conjugate, DGLsPEG, was purified by ultrafiltration, and the buffer was changed to PBS (pH 7.0). Then DGLs-PEG was reacted with YIGSR peptide (sequence: CYGGGYIGSR, Y for short) and EPRNEEK peptide (sequence: CYGGGEPRNEEK, E for short), with a mole ratio of 1:2 (mol/mol, DGLs to peptides) in PBS (pH 7.0) for 24 h at room temperature. The MAL groups of DGLs-PEG were specifically reacted with the thiol groups of the two peptides, yielding the two DGLs-PEG-peptide vectors.21 After purifying by ultrafiltration using a 5 kDa molecular weight cutoff membrane, the characteristics of DGLs-PEG-peptide vectors were analyzed by nuclear magnetic resonance (NMR) spectroscopy. Basically, DGLs-PEG-peptide vectors were solubilized in D2O and analyzed in a 400 MHz spectrometer (Varian, Palo Alto, CA, USA). For the synthesis of BODIPY-labeled vectors, DGLs were first reacted with BODIPY in 100 mM NaHCO3 for 1 h at room temperature, and purified by ultrafiltration using a 5 kDa molecular weight cutoff membrane to remove unreacted BODIPY. The BODIPY-labeled DGLs were used to synthesize different BODIPY-labeled vectors as described above. DGLs derivatives (DGLs-PEG, DGLs-PEG-Y, DGLs-PEG-E) were freshly prepared and diluted to appropriate concentrations in PBS (pH 7.4). DNA solution (100 μg DNA/mL 50 mM sodium sulfate solution) was added to obtain specified weight ratio (6:1, DGLs to DNA, w/w) and immediately vortexed for 30 s at room temperature. Freshly prepared NPs were used in the following experiments. For synthesis of EMA-labeled DNA, fresh plasmid DNA solution (1 mg/mL in TE buffer, pH 7.0) was diluted to 0.1 mg/mL with aqueous solution of EMA and incubated for 30 min in the dark. The complex was then exposed to UV light (365 nm) for 1 h, and the resulting solution was precipitated by adding ethanol to a final concentration of 30% (v/v). The precipitate was collected by centrifugation and redissolved in 50 mM sodium solution. 2.4. Characterization of the DGLs-PEG-peptide/DNA NPs. The morphology of the BBB-targeted NPs (DGLs-PEGY/DNA and DGLs-PEG-E/DNA) as well as DGLs-PEG/DNA was analyzed by transmission electron microscopy (JEM-2010/ INCA OXFORD). The particle sizes and zeta-potential of the

vectors with long circulation and targeting properties. Thus, an efficient targeting ligand is important in brain-targeted gene delivery by DGLs vectors. Recent advances in the gene therapy areas of RNA interference (RNAi) offer a great opportunity to develop molecular targeted therapies in cancer treatment. However, the poor stability, the low delivery efficiency, and the high cost of producing siRNAs are the major challenges for applying siRNAs in cancer therapy. Young-Seok Cho18 has constructed a specially designed siRNA-generating DNA cassette, survivincassette, which includes a U6 promoter in the sequence. After delivery into the cells, the survivin-cassette could interact with cellular transcriptional factors in the nucleus and activate transcription of shRNA genes by the U6 promoter in the DNA cassette, and process them into multiple double-stranded siRNAs for targeted gene silencing. To achieve BBB and glioma dual-targeted gene delivery using nonviral synthetic DGL polymers, the short peptide EPRNEEK derived from pneumococcal CbpA was linked to PEG-modified DGLs as active-targeting ligand. In addition, the pentapeptide Tyr-Ile-Gly-Ser-Arg (YIGSR) derived from the laminin b1 chain, which was reported to specifically bind to laminin receptor,19 was also used to conjugate DGL-PEG as a positive control to evaluate the mechanism and active-targeting ability of the novel EPRNEEK peptide. The two DGL-PEG-peptide vectors were complexed with plasmid DNA, respectively, yielding nanoparticles (NPs) in a self-assembling manner. The BCEC laminin receptor binding of the two peptidemodified NPs can be compared to verify whether they have different binding sites on laminin receptors. Moreover, the cellular uptake, in vivo distribution, and gene transfection experiments also revealed a better affinity of EPRNEEK peptide in the BBB and glioma-targeting effect. We further utilize DGLs-PEG-EPRNEEK vehicles condensing the survivincassette, constructing BBB−glioma dual targeting gene delivery nanoparticles for molecular targeted therapy of glioma.

2. MATERIALS AND METHODS 2.1. Materials. Dendrigraft poly-L-lysines (DGLs) (containing 123 primary amino groups, generation 3) were purchased from COLCOM (Montpellier Cedex, France). αMalemidyl-ω-N-hydroxysuccinimidyl polyethylene glycol (NHS-PEG-MAL, MW 3500) and MAL-PEG-NH2 (MW 2000) were obtained from JenKem Technology Co., Ltd. (Beijing, China). The two laminin receptor binding peptides with a cysteine and four amino-acid spacer on each N terminal (sequence: CYGGGYIGSR and CYGGGEPRNEEK) were synthesized by Chinese Peptides Co., Ltd. The BODIPY fluorophore (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, sulfosuccinimidyl ester, sodium salt), 4,6-diamidino-2-phenylindole (DAPI), ethidium monoazide bromide (EMA), YOYO-1 iodide, and LysoTracker Red were purchased from Molecular Probes (Eugene, OR, USA). The red fluorescent protein (RFP) plasmid (Shanghai GeneChem Co., Ltd., China) and pGL3-control vector (Promega, Madison, WI, USA) were purified using QIAGEN Plasmid Mega Kit (Qiagen GmbH, Hilden, Germany). Fetal bovine serum (FBS) and 0.25% (w/v) trypsin solutionwere purchased from Gibco (Tulsa, OK, USA). Lysis buffer RIPA, GAPDH antibody, and survivin antibody were purchased from Beyotime Institute of Biotechnology (Shanghai, China). 2.2. Cell Lines and Animals. Brain capillary endothelial cells (BCECs) were kindly provided by Prof. J. N. Lou (the 3332

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the supernatant was quantified by a Luciferase Assay System (Promega, Madison, WI, USA), and total amount of cellular protein was determined by Bradford assay, respectively. The final light unit data of each sample was calculated by the light unit of each sample measured minus the light unit of blank sample. The results were expressed as light units/mg protein. 2.8. Binding Site Comparison of Two Laminin Receptor Binding Peptide and the Cellular Uptake Inhibition Assay of the Two Targeted NPs. The two peptides were labeled with BODIPY and Cy7 (Fanbo Biochemicals, China) through a bifunctional linker, PEG (MAL-PEG-NHS, MW 2000), with the same ratio of modification, respectively. After that, the fluorescent peptides were purified by ultrafiltration. Then the BCECs grown on sterile glass coverslips were incubated with free Y peptide, E peptide, and Y plus E peptides before the addition of a mixture solution of BODIPY labeled YIGSR peptide and Cy7 labeled EPRNEEK peptide (1:1, mol/mol). The binding process proceeded at 4 °C for 2 h. The cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature and stained with 300 nM DAPI for 10 min at room temperature. After being washed twice with PBS (pH 7.4), coverslips were mounted and observed by a confocal microscope (Leica TCS SP5, Germany). The emission wavelengths were 488 and 633 nm for BODIPY and Cy7, respectively. In the uptake inhibition experiment, the BCECs were either treated with all three NPs at 4 °C or treated with two free peptides before the addition of three NPs at 37 °C, respectively. The uptake inhibition time lasted for 1 h, and cells were washed with PBS and examined under a fluorescence microscope. 2.9. Tumor Implantation. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Fudan University, Shanghai, China. Glioma-bearing mice were prepared by intracranial injection (striatum, 1.8 mm right lateral to the bregma and 3 mm of depth) of 1 × 105 U-87 MG cells suspended in serum-free media into male nude mice with body weight of 20−25 g. At the 18th day, the male nude mice were intraperitoneally administered with fluorescein potassium and imaged via Xenogen IVIS Lumina System coupled with Living Image software (Xenogen, Corp, Alameda, CA). The luciferase intensity was above 3000, which showed a successful tumor formation. 2.10. In Vivo Imaging Analysis. Nude mice were anesthetized by intraperitoneal injection of 10% chloral hydrate. U-87 MG cells (1 × 105 in 4 μL of PBS 7.4) were implanted into the right striatum (1.8 mm right lateral to the bregma and 3 mm of depth) of the mice by using a stereotactic fixation device with mouse adaptor. The three NPs loading EMA-labeled DNA were injected through the tail vein of tumor-bearing nude mice at a dose of 50 μg of DNA/mouse which was fixed based on our previous research. Images were taken by CRI in vivo imaging system (CRI, Woburn, MA, USA) 90 min after injection after mice were anesthetized. Then, the mice were sacrificed by injection of chloral hydrate via tail vein. The principal organs (including brain, heart, kidney, liver, lung, and spleen) were removed. The ex vivo distribution of NPs was compared by CRI in vivo imaging system. 2.11. Qualitative Distribution of Gene Expression in Mouse Brain and Glioma. The DGLs-PEG-Y/DNA, DGLsPEG-E/DNA NPs (6:1, DGLs to DNA, w/w, red fluorescence protein (RFP) plasmid DNA used in this experiment), and

three NPs were measured by Zetasizer Nano (Malven, England) 2.5. Electrophresis of DGLs-PEG/DNA and DGLs-PEGpeptides/DNA NPs and Their DNA Protection Assay. The three NPs including DGLs-PEG/DNA, DGLs-PEG-Y/DNA, and DGLs-PEG-E/DNA were freshly prepared with DGLs to DNA at weight ratio of 6:1. 0.7% agarose gel electrophoresis was performed to evaluate the DNA encapsulation effect caused by DGL in the NPs compared with naked DNA. To determine the NPs stability to enzymes, NcoI and XhoI (Promega, USA) were added to the three NPs, respectively, and the mixtures were incubated at 37 °C for 2 h. The reaction was stopped at 65 °C for 10 min to inactivate the enzymes. After that, 15 mg/ mL sodium heparin was added and incubated at room temperature for 2 h to release the DNA from the NPs. All the samples were analyzed by 0.7% agarose gel electrophoresis. The integrity of the plasmid in each sample was compared with untreated naked DNA and NcoI and XhoI treated naked DNA. 2.6. Qualitative and Quantitative Evaluation of DGLs Derivatives/DNA NPs Cellular Uptake by BCECs and U87 MG Cells. The BCECs and U-87 MG cells were respectively seeded at a density of 2 × 104 cells/well in 24well plates (Corning-Coaster, Tokyo, Japan), incubated for 72 h, and checked under the microscope for similar confluency and morphology. After this, BCECs and U-87 MG cells were incubated with DGLs-PEG/DNA, DGLs-PEG-Y/DNA, or DGLs-PEG-E/DNA NPs loading EMA labeled DNA at the concentration of 30 μg/well measured by DGLs in the DMEM for 1 h at 37 °C. Then, cells were washed with PBS (pH 7.4) three times and observed by fluoresce microscope (Leica, Germany). In the case of flow cytometry analysis, BCECs and U-87 MG cells were seeded at a density of 10 × 104 cells/well in 6-well plates (Corning-Coaster, Tokyo, Japan), incubated for similar confluency and morphology. The cells were incubated with three BODIPY-labeled NPs for 60 min. The cells were washed three times with phosphate buffer solution (PBS, pH 7.4), trypsinized, and centrifuged at 1200 rpm for 5 min to obtain a cell pellet, which was subsequently resuspended in PBS (pH 7.4) and analyzed using a flow cytometer (BD, USA). The fluorescence of BODIPY was collected at 520 nm (FITCchannel). For each sample, 10,000 events were collected and analyzed. Cells cultured under the normal conditions served as the control. 2.7. Qualitative and Quantitative Evaluation of DGLs Derivatives/DNA NPs Gene Transfection by U-87 MG Cells. U-87 MG cells were seeded at a density of 5 × 104 cells/ well in a 24-well plate and grown to reach 70−80% confluence prior to transfection. Before transfection, the medium was exchanged with fresh serum-free medium. The cells were treated with different NPs solutions containing 5 μg of plasmid EMA labeled DNA for 4 h at 37 °C. After exchange with a fresh serum-containing medium, cells were further incubated for 2 days after transfection. Five micrograms of plasmid DNA mixed with Lipofectamine2000 according to the standard protocol as described in instructions served as positive control. In the case of qualitative evaluation, the red fluorescence images were taken using a fluorescence microscope. For luciferase activity assay, medium was removed and the cells were rinsed with PBS (without calcium) and shaken for 30 min at room temperature in 150 μL of luciferase cell culture lysis reagent supplied by the Promega Luciferase Assay Kit. The lysis solution was centrifuged at 14000g for 2 min at 4 °C. Luciferase activity in 3333

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Figure 2. Characterization of DGLs-PEG/DNA and two DGLs-PEG-peptide/DNA nanoparticles. NMR spectra of (A) DGLs-PEG-Y and (B) DGLs-PEG-E in D2O at 400 MHz. (C) Particle sizes and zeta-potential of DGLs-PEG/DNA, DGLs-PEG-Y/DNA, and DGLs-PEG-E/DNA NPs. TEM images of (D) DGLs-PEG-Y/DNA NPs and (E) DGLs-PEG-E/DNA NPs. (F) Agarose gel electrophoresis evaluation of DNA encapsulation and protection of NPs. Lane 1: marker. Lane 2: naked DNA. Lane 3: DGLs-PEG/DNA NPs. Lane 4: DGLs-PEG-Y/DNA NPs. Lane 5: DGLs-PEGE/DNA NPs. The stability of NPs loading DNA against enzyme digestion. Plasmid DNA was released from the NPs by the addition of sodium heparin separated by agarose gel electrophoresis after enzymes incubation. Lane 6: naked plasmid DNA treated with enzymes. Lanes 7−9: DGLsPEG/DNA, DGLs- PEG-Y/DNA, and DGLs-PEG-E/DNA NPs with treatment of heparin after enzyme incubation. 3334

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forward primer was synthesized by Hanbio, Ltd. (Shanghai, China). 2.16. Statistical Analysis. The data are presented as mean ± SD. The statistical significance was determined using Student’s t test and analysis of variance (ANOVA).

DGLs-PEG/DNA NPs were injected through the tail vein of tumor-bearing mice at a dose of 50 μg of DNA/mouse. About 48 h later, animals were anesthetized with 10% chloral hydrate and perfused transcardially with saline followed by 4% paraformaldehyde in PBS. The brains were rapidly removed and postfixed for 24 h, then transferred to PBS containing 30% sucrose at 4 °C until subsidence. Coronal brain sections were made at a thickness of 30 μm with a cryotome Cryostat (Leica, CM 1900, Wetzlar, Germany) and stained with 300 nM DAPI for 10 min at room temperature. After washing twice with PBS (pH 7.4), the sections were immediately observed under the fluorescence microscope. 2.12. Quantitative Expression of Reporter Gene in Vivo. The three NPs (6:1, DGLs to DNA, w/w, pGL-3 control plasmid used in this experiment) were injected into the tail vein of tumor-bearing mice at a dose of 50 μg of DNA/mouse. At 48 h after injection, the mice were sacrificed by injection of chloral hydrate via tail vein and the principal organs (including brain, heart, liver, lung, and kidney) were extirpated. The organs were carefully washed with distilled water and homogenized in 1 mL of lysis reagent (Promega, Madison, WI, USA) using a JY92IIN tissue homogenizer (Scientz, China). The homogenate was centrifuged at 14000g for 20 min at 4 °C. Luciferase activity and total proteins in the supernatant were measured similarly to the cellular experiment as previously described. 2.13. In Vivo Pharmacodynamic Evaluation and Survival Monitoring. The glioma-bearing mice were randomized to four groups (10 mice/group). At the 12th, 15th, and 18th days after the implantation, each group of mice was treated with intravenous administration of saline, DGLsPEG/survivin NPs, DGLs-PEG-E/scramble NPs, and DGLPEG-E/survivin NPs at a dose of 50 μg of DNA/mouse. At the 21st day, the mice were monitored through Xenogen IVIS Lumia System coupled with Living Image software (Xenogen, Corp, Alameda, CA) for pharmacodynamics evaluation. 2.14. Western Blot Analysis. The glioma-bearing mice were randomized to four groups (3 mice/group) and treated at the 12th, 15th, and 18th days after the implantation as previously. At the 21st day, the mice were sacrificed and the glioma tissue was lysed using lysis buffer RIPA at a concentration of 10 μL/mg. A total of 50 μg of proteins were resolved on 12% polyacrylamide−SDS gels and then transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in Tris-buffer saline for 1 h, and incubated overnight with primary antibodies for survivin and GAPDH. After three washes, the membranes were incubated with anti-rabbit or anti-mouse secondary antibodies conjugated with horseradish-peroxidase for 1 h. The levels of specific proteins in each lysate were detected by enhanced chemiluminescence using ECL plus followed by autoradiography. 2.15. RT-PCR for Evaluating Survivin mRNA. The glioma-bearing mice were randomized to four groups (3 mice/group) and treated at the 12th, 15th, and 18th days after the implantation as previously. At the 21st day, the mice were sacrificed and the glioma tissues were excised for extraction of total RNA. In vivo expression of survivin mRNA was detected by RT-PCR. Total RNA was extracted by using TRIzol reagent, and possible DNA contamination was removed by digesting the extracted RNA with DNase I. The RNA was purified again using TRIzol reagent and subjected to the synthesis of firststrand cDNA using a reverse transcription kit. GADPH was amplified as an internal control. The sequence of the survivin

3. RESULTS AND DISCUSSION The delivery of an exogenous therapeutic gene into the glioma by systemic administration becomes difficult because of the BBB. Considering the overexpression of laminin receptors in glioma cell lines and in the BBB, designing the laminin receptor

Figure 3. Qualitative and quantitative evaluation of DGLs derivatives/ DNA NPs cellular uptake by BCECs and U-87 MG cells. Fluorescent images of BCECs uptake after the incubation of (A) DGLs-PEG/ DNA, (B) DGLs-PEG-Y/DNA, and (C) DGLs-PEG-E/DNA NPs loading EMA-labeled DNA for 1 h. Original magnification: ×200. (D) Comparison of mean fluorescent intensities in BCECs treated with BODIPY-labeled DGLs-PEG/DNA, DGLs-PEG-Y/DNA, and DGLsPEG-E/DNA NPs for 1 h. Fluorescent images of U-87 MG cells uptake after the incubation of (E) DGLs-PEG/DNA, (F) DGLs-PEGY/DNA, and (G) DGLs-PEG-E/DNA NPs loading EMA-labeled DNA for 1 h. Original magnification: ×200. (H) Comparison of mean fluorescent intensities in U87 MG cells treated with BODIPY-labeled DGLs-PEG/DNA, DGLs-PEG-Y/DNA, and DGLs-PEG-E/DNA NPs for 1 h. **, p < 0.005; ***, p < 0.001, significance represents comparison between two groups. 3335

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reagent was used to determine the percentage of unreacted thiol. And little thiol was detected (data not shown) which could be explained as the thiol at the end of peptides reacted with the MAL at one end of PEG specifically. The three DGLs derivatives/DNA (DGLs-PEG/DNA, DGLs-PEG-YIGSR/ DNA, and DGLs-PEG-EPRNEEK/DNA; DGLs-PEG/DNA, DGLs-PEG-Y/DNA, and DGLs-PEG-E/DNA for short, respectively) NPs were freshly prepared before use. As shown in Figure 2C, the sizes of the three NPs were all less than 120 nm measured by dynamic light scattering (DLS). The two peptide-modified NPs were slightly larger than the unmodified ones, which may be explained as the peptide modification might increase steric hindrance when DGLs derivatives were complexed with plasmid DNA. The TEM results (Figure 2D,E) confirmed that the peptide-modified NPs were spherical and homogeneous particles with a diameter of 90 nm. It was reasonable that the size measured by DLS was larger than that measured by TEM because the DLS analysis indicated the hydrated value of the particle size. The size below 120 nm was thought to be suitable enough for BBB-targeted drug delivery systems as the size of brain capillary was smaller than that of other capillaries. The zeta-potential results showed that all three NPs were positively charged with a surface charge of 3 mV. The positive charge was mainly attributed to the protonated amino group at the surface of DGLs. This result also proved that DGLs could effectively encapsulate the plasmid to neutralize the negative charge of DNA. The positive charge may favor the binding with cell membrane, which could facilitate the adsorptive-mediated endocytosis. Figure 2F showed the electrophoresis results of DGLs derivatives/DNA NPs. All three NPs with DGLs to DNA at weight ratio of 6:1 could encapsulate DNA completely with no electrophoresis shift (Figure 2F lanes 3−5) compared with naked plasmid DNA (Figure 2F lane 2). After treatment with two restriction enzymes which could recognize two specific sequences in the plasmid yielding the DNA dividing into two fragments, all three NPs were incubated with heparin which was negatively charged and could bind to DGLs, resulting in the DNA releasing from the NPs. The integrity maintained in all three groups compared with naked plasmid DNA. This result demonstrated that the DGLs derivatives could play their DNA protection effect before uptake of NPs by cells and the stability of the NPs was enough to resist the enzymes in surrounding circumstances. 3.2. Cellular Uptake of the NPs by BCECs and U-87 MG Cells. BCECs were incubated with all three NPs for 60 min. Fluorescent intensity was compared by both observation under the microscope (Figure 3A−C) and the flow cytometry measurement (Figure 3D). In the case of fluorescent imaging, the signal of cells treated with DGLs-PEG-E/DNA NPs loading EMA-labeled DNA was the highest, while the cellular uptake of DGLs-PEG-Y/DNA NPs was less but still higher than that of DGLs-PEG/DNA NPs. Flow cytometry analysis provided the quantitative result which could verify the results observed in fluorescent images. Among the BODIPY-labeled DGLs derivatives/DNA NPs, the fluorescent intensity of DGLsPEG-E/DNA NPs was still the highest. Both qualitative evaluations observed by microscope and quantitative evaluations by flow cytometry analysis were consisted with the previous study without the presence of serum (data not shown) showing that the stability and uptake characteristics of the NPs

Figure 4. Qualitative and quantitative evaluation of DGLs derivatives/ DNA NPs gene transfection by U-87 MG cells. The qualitative (A−D) and quantitative (E) evaluation of gene transfection efficiency in vitro. The fluorescence images of RFP expression in U-87 MG cells were taken 48 h post-transfection with (A) DGLs- PEG/DNA, (B) DGLsPEG-Y/DNA, (C) DGLs-PEG-E/DNA NPs, and positive control (D) Lipofectamine2000/DNA NPs. Red: RFP. Original magnification: ×200. Luciferase activity was measured 48 h post-transfection and expressed as light units per mg protein. Data represent the mean ± SEM (n = 4). **, p < 0.005, compared between two groups.

binding peptide can have dual-targeted effects on the BBB and glioma cells in early stage glioma and initiate prompt intervention for brain tumor. EPRNEEK derived from the pneumococcal CbpA domain had a key function in guiding Streptococcus pneumonia across the BBB, causing meningitis.22 Herein, constructing EPRNEEK modified DGL-PEG as templates for gene delivery may overcome the hurdle of the BBB and achieve a desired therapeutic effect of glial tumors. Another YIGSR peptide derived from endogenous substances was used in this study for comparison because both peptides bind to laminin receptors in the BBB. In the therapeutic study, we used a specially designed DNA cassette survivin, which could interact with cellular transcriptional factors in the nucleus and activate transcription of shRNA genes after transport into tumor cells. Then the shRNA could be processed into double-stranded siRNAs for silencing survivin protein and achieving ideal antitumor efficacy. 3.1. Characterization of DGLs-PEG/DNA and Two DGLs-PEG-peptide/DNA Nanoparticles. The NMR spectral results (Figure 2A,B) proved the existence of the conjugate structures of DGLs-PEG-peptide vectors. Meanwhile Ellman’s 3336

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Figure 5. Binding sites analysis. Confocal images of the two peptides binding to BCECs membrane. (A, E) No treatment, (B, F) free YIGSR peptide, (C, G) EPRNEEK peptide, and (D, H) YIGSR plus EPRNEEK peptides incubation before the addition of a mixture solution of BODIPY labeled YIGSR peptide and Cy7 labeled EPRNEEK peptide (1:1, mol/mol) at 4 °C. Green: BODIPY labeled YIGSR peptide. Red: Cy7 labeled EPRNEEK peptide. Blue: DAPI labeled cell nuclei. (A−D) Merged images of green signal and red signal. (E−H) Merged images of three fluorescent signals and bright field.

laminin receptor. The two peptides were both found to bind to the membrane. With the addition of free YISGR peptide, the red signal remained at the same level as for untreated cells, while the green signals could not be detected any more (Figure 5B,F). As shown in Figure 5C,G, only the YISGR peptide was observed to bind to the cell membrane. This result illustrated that free YISGR peptide could not affect the binding between EPRNEEK peptide and laminin receptor. The binding of YISGR peptide with laminin receptor could not be inhibited by free EPRNEEK peptide. Furthermore, the binding was not observed when BCECs were treated with both free YISGR peptide and EPRNEEK peptide (Figure 5D,H). The results demonstrated that the two peptides had different binding sites on laminin receptor which could not be interfered with by each other. However, EPRNEEK peptide showed a better affinity than YISGR peptide, which resulted in the better cellular uptake and gene expression ability presented before. Therefore, we chose EPRNEEK peptide modified vehicle, DGLs-PEG-E/ DNA NPs, for the in vivo evaluation of therapeutic antitumor efficacy. 3.5. In Vivo Distribution of NPs. The tumor-bearing nude mice were injected with the nontargeted DGLs-PEG/DNA or two DGLs-PEG-peptide/DNA NPs loading a fluorescent probe EMA-labeled DNA, respectively. In vivo fluorescent images were taken at 90 min after injection. As shown in Figure 6A, EMA-labeled DNA was obviously accumulated in brain in the tumor-bearing mice treated with the DGLs-PEG-E/DNA NPs, while the fluorescence in the brain of the DGLs-PEG-Y/DNA NPs treated nude mice was less. This difference is partially attributed to the different binding sites of EPRNEEK and YISGR. DGLs-PEG-Y/DNA NPs might be interrupted by endogenous laminins, while DGLs-PEG-E/DNA NPs is less likely to be interrupted. In the case of DGLs-PEG/DNA NPs, the fluorescent signal was not so significant, because without the help of active-targeting ligand, NPs cannot penetrate the BBB spontaneously. The ex vivo organ distribution (Figure 6B) revealed the increasing uptake of DGLs-PEG-E/DNA NPs in the brain, especially at the glioma site. The fluorescence was also observed in heart, liver, lung, and kidney. The accumulation in liver

were not affected by serum. These results indicated that the NPs had the potential for in vivo application further. The EMA-labeled DGLs derivatives/DNA NPs were used to investigate cellular uptake characteristics in U-87 MG cells. The results were shown qualitatively using fluorescent images (Figure 3E−G). The fluorescence intensity exhibited by U-87 MG cells had increased when cells were treated with the DGLsPEG-Y/DNA and DGLs-PEG-E/DNA NPs loading EMAlabeled DNA compared with DGLs-PEG/DNA NPs. The quantitative result presented by flow cytometry analysis confirmed the qualitative fluorescence results (Figure 3H). The BODIPY-labeled DGLs were used in this experiment. The mean fluorescent intensities of cells incubated with DGLs-PEGE/DNA NPs were significant higher than that incubated with DGLs-PEG-Y/DNA and DGLs-PEG/DNA NPs. 3.3. In Vitro Gene Transfection. The transfection efficiency mediated by DGLs series vector/DNA NPs was assessed in U-87 MG cells. Figure 4A−D gave a qualitative comparison of the DGLs-PEG/DNA, DGLs-PEG-Y/DNA, and DGLs-PEG-E/DNA NPs as well as positive control lipofectamine2000/DNA. The RFP expression of DGLs-PEG-Y/DNA, DGLs-PEG-E/DNA NPs and lipofectamine2000, the positive control, were much higher than that of DGLs-PEG/DNA NPs. The luciferase activity of cells treated with DGLs-PEG-Y/DNA, DGLs-PEG-E/DNA NPs, and Lipofectamine2000 was also higher than that of DGLs-PEG/DNA NPs (Figure 4E). 3.4. Binding Sites Analysis. Considering the different cellular uptake ability and gene expression ability induced by EPRNEEK and YISGR, we hypothesized that the binding sites of the two active-targeting peptides are the cause of that. To reveal the cellular uptake difference of the two peptide-modified NPs, the binding sites of the two peptides with laminin receptor were compared. When the BCECs were incubated with a mixture of the two peptides at the mole ratio of 1 to 1, the colocation was observed as the yellow spots were overlay signals from green fluorescence labeled YISGR peptide and red fluorescence labeled EPRNEEK peptide (Figure 5A,E). This figure also revealed that EPRNEEK peptide could bind more to cell membrane compared with YISGR peptide which indicated that EPRNEEK peptide might have a higher binding affinity to 3337

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Figure 7. Distribution of RFP gene expression in brains of tumorbearing mice treated with DGLs-PEG/DNA NPs (A, D, G, and J), DGLs-PEG-Y/DNA NPs (B, E, H, and K), and DGLs-PEG-E/DNA NPs (C, F, I, and L) 48 h after iv administration. Frozen sections (30 μm thick) of caudate putamen (A−C), hippocampus (D−F), cortical layer (G−I), and tumor site (J−L) were examined by fluorescent microscopy. The sections were stained with 300 nM DAPI for 10 min at room temperature. Red: RFP. Blue: DAPI stained cell nuclei. Original magnification: ×200.

Figure 6. In vivo distribution of DGLs derivatives/DNA NPs. (A) In vivo fluorescence images of animals at 90 min after intravenous injection of DGLs-PEG/DNA, DGLs-PEG-Y/DNA, and DGLs-PEGE/DNA NPs (from left to right). (B) Ex vivo fluorescence images of organs harvested 90 min after DGLs-PEG/DNA, DGLs-PEG-Y/DNA, and DGLs- PEG-E/DNA NPs injection (from top to bottom) where B, H, Li, S, Lu, and K represent the brain, heart, liver, spleen, lungs, and kidney, respectively. (C and D) Coronal section of tumor site. Fluorescence signal was from EMA-labeled DNA.

decreased in mice treated with DGLs-PEG-Y/DNA and DGLsPEG-E/DNA NPs compared to that treated with DGLs-PEG/ DNA NPs, because with PEGylation modification, nanoparticles can present a prolonged blood circulation and reduced capture by the reticuloendothelial system23−25 and also increase the possibility of binding between active-targeting ligands and their receptors in the BBB, which contributes to the brain and glioma site uptake. The DGLs-PEG-Y/DNA NPs were observed to possess the most retention in lungs. This result was in accordance with previous study findings on the YIGSR peptide, which has been widely applied in lung-targeted drug delivery systems.26 The results also showed that the three NPs were found in the kidneys (Figure 6B), suggesting that the kidney is a principal pathway responsible for NP clearance. The fluorescent signals were more obvious in the tumor site of the one treated with DGLs-PEG-E/DNA NPs (Figure 6C,D). All

Figure 8. Quantitative evaluation of gene expression in vivo. Luciferase expression 48 h after iv administration of DGLs-PEG/DNA, DGLsPEG-Y/DNA, and DGLs-PEG-E/DNA NPs in tumor-bearing mice at a dose of 50 μg of pGL-3 control plasmid DNA/mouse. Luciferase expression of (A) brain and tumor and (B) other principal organs is plotted as light units per mg protein. Data are expressed as mean ± SEM (n = 4). **, p < 0.01; ***, p < 0.001, significance represents comparison between two groups.

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Figure 9. Antitumor efficacy. (A) Relative enhancement of luciferase signals in luci-U87 glioma-bearing mice after treatment (n = 6). (B) In vivo inhibition of endogenous survivin mRNA expression by RT-PCR. (C) Western blot analysis. In vivo inhibition of survivin protein expression after treatment. (B, C) Lane 1: saline. Lane 2: DGLs-PEG/survivin. Lane 3: DGLs-PEG-E/survivin. Lane 4: DGLs-PEG-E/scramble. GADPH was used as an internal control. **, p < 0.01; ***, p < 0.001, significance represents comparison between two groups. (D) Overall survival of glioma-bearing mice (n = 10).

3.7. Quantitative Analysis of Gene Expression in Vivo. The transfection efficiencies of DGLs-PEG/DNA, DGLs-PEGY/DNA, and DGLs-PEG-E/DNA NPs loading pGL-3 control plasmid in principal organs including the tumor were measured after 48 h (Figure 8). The luciferase activity of the DGLs-PEGE/DNA NPs in the brain was over 1-fold higher than that of the DGLs-PEG/DNA NPs. Meanwhile, the luciferase activity of brain tissues treated with DGLs-PEG-Y/DNA was less than that of brain tissues treated with DGLs-PEG-E/DNA NPs and higher compared with DGLs-PEG/DNA NPs (Figure 8A). The luciferase activity of the tumor from DGLs-PEG-E/DNA NPs treated mice was the most among all three groups, which was the same tendency in the distribution results. The luciferase expression of the DGLs-PEG-E/DNA NPs in the lung, liver, and kidney was declined compared with that of

of the results indicated that DGLs-PEG-E/DNA NPs possessed good tumor target ability and internalization efficiency. 3.6. Qualitative Analysis of Distribution of Gene Expression in Brain and Tumor Site. RFP expression in the different parenchyma areas including cortical layer, hippocampus, caudate putamen, and the tumor site at 48 h after administrated DGLs-PEG/DNA, DGLs-PEG-Y/DNA, or DGLs-PEG-E/DNA NPs are shown in Figure 7A−L. The gene expression in the four regions treated with DGLs-PEG/DNA NPs was less than that treated with peptide-modified NPs. For the DGLs-PEG-E/DNA NPs, gene expression observed in the tumor site (Figure 7L) and hippocampus (Figure 7F) was higher than that of the DGLs-PEG-Y/DNA NPs, especially. This is also partially because of the different binding affinities of EPRNEEK and YISGR. 3339

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Binding site analysis revealed that the difference between the two peptide-modified NPs was attributed to their different binding sites on the laminin receptor. A significant increase in gene expression was observed both in vitro and in vivo, with the administration of targeted NPs. In vivo pharmacodynamics evaluation exhibited a prolonged survival time in DGLs-PEGE/survivin NPs, which revealed that the capability of the activetargeting nanoparticle carrier for simultaneous delivery of therapeutic agents (survivin-cassette) might enhance the effectiveness of glioma therapy. In summary, our study suggested that the EPRNEEK peptide derived from pneumococcal CbpA provided an effective modification for DGL-based DNA-loaded NPs to improve brain glioma dual-targeted gene delivery.

DGLs-PEG/DNA NPs, whereas heart levels were not changed markedly (Figure 8B). The luciferase activity in lung with DGLs-PEG-Y/DNA NPs injection was the highest, which confirmed the lung-targeted characteristic of YIGSR peptide applied in several studies. Considering the potential activetargeting efficiency of EPRNEEK, we chose EPRNEEK peptide as BBB−glioma dual targeting ligand for the following in vivo pharmacodynamics evaluation. 3.8. Antitumor Efficacy. The clinically therapeutic benefits are mainly determined based on the quality of life and prolonged survival time of cancer patients.27 Considering the better BBB−glioma dual targeting efficiency of EPRNEEK, we used DGLs-PEG-E vehicles as active-targeting gene delivery templates. Saline was used as control group and a scramble DNA was used as control DNA with no therapeutic effect. Survivin-cassette was used as antitumor gene drug for the treatment. The relative enhancement of luciferase signal intense was measured, which represented the growth of tumor. After 3 times’ treatment, the DGLs-PEG-E/survivin group showed a significant glioma curing effect with an inhibited luciferase signal comparing to that of saline, DGLs-PEG/survivin, and DGLs-PEG-E/scramble NPs, which represented a reduced tumor growth (Figure 9A). To further evaluate the antitumor efficacy, the overall survival of the glioma-bearing mice was estimated (Figure 9D). The control group (saline) exhibited an early death as a function of time, while DGLs-PEG-E/survivin groups showed a prolonged survival time (Figure 9D). This is mainly attributed to the dual targeting effect of DGLs-PEG-E/ survivin NPs and the survivin-cassette. After the internalization of DGLs-PEG-E/survivin NPs, survivin-cassette could be released into cytoplasm and enter into the cell nucleus. The U6 promoter of the survivin-cassette can interact with cellular transcriptional factors and then activate transcription of shRNA genes, which can be processed into double-stranded siRNA for targeted gene silencing of survivin, which is an anti-cell death gene that confers resistance of cancer cells to therapeutic agents.28,29 RT-PCR analysis was performed to evaluate the level of survivin mRNA (Figure 9B). The survivin mRNA of in vivo glioma was remarkably inhibited in the group of DGLs-PEG-E/ survivin NPs because of the successful internalization of DGLsPEG-E/survivin NPs and the transcription of survivin-siRNA from survivin-cassette. The Western blot analysis also verified the efficient delivery of DNA-cassettes and the pharmacological effects of survivincassette. The level of survivin proteins was markedly downregulated by the DGLs-PEG-E/survivin NPs, while the survivin proteins remained almost the same in saline, DGLs-PEG/ survivin NPs, and DGLs-PEG-E/scramble NPs groups (Figure 9C). Therefore, DGLs-PEG-E/survivin NPs showed a great tumor-targeting ability and an ideal potential for antitumor therapy in vivo pharmacodynamic evaluation.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-21-5198-0079. Fax: +86-21-5198-0079. E-mail: [email protected]. Author Contributions ‡

Y.L. and X.H. contributed equally to this manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from National Basic Research Program of China (973 Program, 2013CB932500), National Natural Science Foundation of China (81373355), and Program for New Century Excellent Talents in University.

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REFERENCES

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4. CONCLUSIONS In this study, two peptides (YIGSR and EPRNEEK) derived from laminin and pneumococcal CbpA, respectively, were used to modify PEGylated DGLs yielding the laminin receptortargeted gene vector in the BBB and the glioma. The data collected in this study indicated that the EPRNEEK peptidemodified NPs were not only more preferable for uptake by both BCECs and U-87 MG cells but also accumulated in the glioma more efficiently than the YIGSR peptide-modified NPs. 3340

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