A Bacteria Deriving Peptide Modified Dendrigraft Poly‑L‑lysines(DGL) Self-Assembling Nanoplatform for Targeted Gene DeliveryYang 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 gliomadepends on gene delivery systems. The gene delivery systemshould be able to cross the blood−brain barrier (BBB) andfurther target glioma at its early stage. Active brain tumortargeted delivery can be achieved using the “Trojan horse”technology, which involves either endogenous ligands orextraneous substances that can recognize and bind to specificreceptors in target sites. This method facilitates receptor-mediated endocytosis to cross the BBB and enter into gliomacells. Dendrigraft poly-L-lysines (DGLs), which are novelnonviral gene vectors, are conjugated to a peptide (sequence:EPRNEEK) derived from Streptococcus pneumonia, a pathogencausing meningitis. This process yields peptide-modifiednanoparticles (NPs) after DNA loading. Cellular uptake and in vivo imaging results indicate that EPRNEEK peptide-modifiedNPs have a better brain tumor targeted effect compared with a pentapeptide derived from endogenous laminin after intravenousinjection. The mechanism of this effect is further explored in the present study. Besides, EPRNEEK peptide-modified NPs alsoexhibited a prolonged median survival time. In conclusion, the EPRNEEK peptide-modified DGL NPs exhibit potential as anonviral 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 centralnervous 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 differentstages. Thus, different strategies should be applied in designinga drug delivery system for targeting glioma. In the treatment oflow-grade glioma, the drug delivery system should be able tocross the blood−brain barrier (BBB) and further target glialcancer cells.2 However, drugs especially for macromolecularproteins and genes are excluded from the brain whenadministered intravenously because of the BBB, which isformed mainly by brain capillary endothelial cells (BCECs).3

Much research has been done to achieve a desirable delivery ofdrugs to glioma by using active-targeting ligands, such astransferrin,4 dehydroascorbic acid (DHA),5 and angiopep-2.6 Inaddition to these endogenous substances, certain pathogens cancross the BBB in a transcellular or paracellular manner andcause CNS infection.7 Various infectious agents, includingprions and certain neurotropic viruses, bind to the lamininreceptor, thereby determining tropism for the CNS.8 Theexpression of laminin receptors is increased in most adultneurons as well as glial cells.9 Laminin receptors also have animportant function in tumor invasion and metastasis.10 The

above evidence indicates that laminin receptors can furtherfacilitate the laminin receptor ligands or their modified drugdelivery system accumulating in the glioma after crossing theBBB. Laminin receptors are found to initiate bacterial contactwith the BBB in experimental meningitis models.11 The criticalsequence enabling the pneumococcal CbpA domain to bind tothe laminin receptor is the sequence EPRNEEK. Consideringthe important function of binding to the laminin receptor ininitiating intimate contact between the circulating bacterialmeningeal pathogens and the BBB cells,9,12 we hypothesize thatthe short peptide (sequence: EPRNEEK) favors lamininreceptor binding in the BBB. This process further results inpreferable BBB translocation and glioma-targeted accumula-tion.Viral gene vectors have been proven efficient; however, safety

concerns have somehow prevented further applications.13

Special Issue: Recent Molecular Pharmaceutical Development inChina

Received: January 28, 2014Revised: May 4, 2014Accepted: June 25, 2014

Article

pubs.acs.org/molecularpharmaceutics

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Synthetic polymers used in nonviral approaches have beeninvestigated intensively. Polyamidoamine (PAMAM)14 andpoly(ether imide) (PEI)15 have shown their potential in genedelivery because of their high encapsulation efficiency.However, the high cytotoxicity drawn by their poordegradability has enormously confined their application.

Dendrigraft poly-L-lysines (DGLs), a new kind of syntheticpolymers consisting of lysine,16,17 have been employed as agene vector because of their degradability and rich externalamino groups that can encapsulate plasmid DNA throughelectric interactions. They can also be modified with poly-ethylene glycol (PEG) and targeting ligands, thereby rendering

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

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vectors with long circulation and targeting properties. Thus, anefficient targeting ligand is important in brain-targeted genedelivery by DGLs vectors.Recent advances in the gene therapy areas of RNA

interference (RNAi) offer a great opportunity to developmolecular targeted therapies in cancer treatment. However, thepoor stability, the low delivery efficiency, and the high cost ofproducing siRNAs are the major challenges for applyingsiRNAs in cancer therapy. Young-Seok Cho18 has constructed aspecially designed siRNA-generating DNA cassette, survivin-cassette, which includes a U6 promoter in the sequence. Afterdelivery into the cells, the survivin-cassette could interact withcellular transcriptional factors in the nucleus and activatetranscription of shRNA genes by the U6 promoter in the DNAcassette, and process them into multiple double-strandedsiRNAs for targeted gene silencing.To achieve BBB and glioma dual-targeted gene delivery using

nonviral synthetic DGL polymers, the short peptide EPRNEEKderived from pneumococcal CbpA was linked to PEG-modifiedDGLs as active-targeting ligand. In addition, the pentapeptideTyr-Ile-Gly-Ser-Arg (YIGSR) derived from the laminin b1chain, which was reported to specifically bind to lamininreceptor,19 was also used to conjugate DGL-PEG as a positivecontrol to evaluate the mechanism and active-targeting abilityof the novel EPRNEEK peptide. The two DGL-PEG-peptidevectors were complexed with plasmid DNA, respectively,yielding nanoparticles (NPs) in a self-assembling manner.The BCEC laminin receptor binding of the two peptide-modified NPs can be compared to verify whether they havedifferent binding sites on laminin receptors. Moreover, thecellular uptake, in vivo distribution, and gene transfectionexperiments also revealed a better affinity of EPRNEEK peptidein the BBB and glioma-targeting effect. We further utilizeDGLs-PEG-EPRNEEK vehicles condensing the survivin-cassette, constructing BBB−glioma dual targeting gene deliverynanoparticles for molecular targeted therapy of glioma.

2. MATERIALS AND METHODS2.1. Materials. Dendrigraft poly-L-lysines (DGLs) (con-

taining 123 primary amino groups, generation 3) werepurchased from COLCOM (Montpellier Cedex, France). α-Malemidyl-ω-N-hydroxysuccinimidyl polyethylene glycol(NHS-PEG-MAL, MW 3500) and MAL-PEG-NH2 (MW2000) were obtained from JenKem Technology Co., Ltd.(Beijing, China). The two laminin receptor binding peptideswith a cysteine and four amino-acid spacer on each N terminal(sequence: CYGGGYIGSR and CYGGGEPRNEEK) weresynthesized by Chinese Peptides Co., Ltd. The BODIPYfluorophore (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-in-dacene-3-propionic acid, sulfosuccinimidyl ester, sodium salt),4,6-diamidino-2-phenylindole (DAPI), ethidium monoazidebromide (EMA), YOYO-1 iodide, and LysoTracker Red werepurchased from Molecular Probes (Eugene, OR, USA). The redfluorescent protein (RFP) plasmid (Shanghai GeneChem Co.,Ltd., China) and pGL3-control vector (Promega, Madison, WI,USA) were purified using QIAGEN Plasmid Mega Kit (QiagenGmbH, Hilden, Germany). Fetal bovine serum (FBS) and0.25% (w/v) trypsin solutionwere purchased from Gibco(Tulsa, OK, USA). Lysis buffer RIPA, GAPDH antibody, andsurvivin antibody were purchased from Beyotime Institute ofBiotechnology (Shanghai, China).2.2. Cell Lines and Animals. Brain capillary endothelial

cells (BCECs) were kindly provided by Prof. J. N. Lou (the

Clinical Medicine Research Institute of the Chinese-JapaneseFriendship Hospital). Primary BCECs were cultured asdescribed previously.20 Cells used in this study were betweenpassage 10 and passage 20. The U-87 MG human glioblastoma-astrocytoma cell line (ATCC number: HTB-14) was kindlyprovided by Prof. W. Y. Lu (School of Pharmacy, FudanUniversity). All cells were cultured at 37 °C under a humidifiedatmosphere 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 fromthe Department of Experimental Animals, Fudan University,and maintained under standard housing conditions. All animalexperiments were carried out in accordance with guidelinesevaluated and approved by the ethics committee of FudanUniversity.

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 withNHS-PEG3500-MAL 1:5 (mol/mol) in PBS (pH 8.0) for 2 hat room temperature. The primary amino groups on the surfaceof DGLs were specifically reacted with the NHS groups of thebifunctional PEG derivative. The resulting conjugate, DGLs-PEG, was purified by ultrafiltration, and the buffer was changedto PBS (pH 7.0). Then DGLs-PEG was reacted with YIGSRpeptide (sequence: CYGGGYIGSR, Y for short) and EPR-NEEK 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 ofDGLs-PEG were specifically reacted with the thiol groups ofthe two peptides, yielding the two DGLs-PEG-peptidevectors.21 After purifying by ultrafiltration using a 5 kDamolecular weight cutoff membrane, the characteristics ofDGLs-PEG-peptide vectors were analyzed by nuclear magneticresonance (NMR) spectroscopy. Basically, DGLs-PEG-peptidevectors were solubilized in D2O and analyzed in a 400 MHzspectrometer (Varian, Palo Alto, CA, USA). For the synthesisof BODIPY-labeled vectors, DGLs were first reacted withBODIPY in 100 mM NaHCO3 for 1 h at room temperature,and purified by ultrafiltration using a 5 kDa molecular weightcutoff membrane to remove unreacted BODIPY. TheBODIPY-labeled DGLs were used to synthesize differentBODIPY-labeled vectors as described above. DGLs derivatives(DGLs-PEG, DGLs-PEG-Y, DGLs-PEG-E) were freshlyprepared and diluted to appropriate concentrations in PBS(pH 7.4). DNA solution (100 μg DNA/mL 50 mM sodiumsulfate solution) was added to obtain specified weight ratio(6:1, DGLs to DNA, w/w) and immediately vortexed for 30 sat room temperature. Freshly prepared NPs were used in thefollowing experiments.For synthesis of EMA-labeled DNA, fresh plasmid DNA

solution (1 mg/mL in TE buffer, pH 7.0) was diluted to 0.1mg/mL with aqueous solution of EMA and incubated for 30min in the dark. The complex was then exposed to UV light(365 nm) for 1 h, and the resulting solution was precipitated byadding ethanol to a final concentration of 30% (v/v). Theprecipitate was collected by centrifugation and redissolved in 50mM sodium solution.

2.4. Characterization of the DGLs-PEG-peptide/DNANPs. The morphology of the BBB-targeted NPs (DGLs-PEG-Y/DNA and DGLs-PEG-E/DNA) as well as DGLs-PEG/DNAwas analyzed by transmission electron microscopy (JEM-2010/INCA OXFORD). The particle sizes and zeta-potential of the

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three NPs were measured by Zetasizer Nano (Malven,England)2.5. Electrophresis of DGLs-PEG/DNA and DGLs-PEG-

peptides/DNA NPs and Their DNA Protection Assay. Thethree NPs including DGLs-PEG/DNA, DGLs-PEG-Y/DNA,and DGLs-PEG-E/DNA were freshly prepared with DGLs toDNA at weight ratio of 6:1. 0.7% agarose gel electrophoresiswas performed to evaluate the DNA encapsulation effect causedby DGL in the NPs compared with naked DNA. To determinethe NPs stability to enzymes, NcoI and XhoI (Promega, USA)were added to the three NPs, respectively, and the mixtureswere incubated at 37 °C for 2 h. The reaction was stopped at65 °C for 10 min to inactivate the enzymes. After that, 15 mg/mL sodium heparin was added and incubated at roomtemperature for 2 h to release the DNA from the NPs. Allthe samples were analyzed by 0.7% agarose gel electrophoresis.The integrity of the plasmid in each sample was compared withuntreated 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 U-87 MG Cells. The BCECs and U-87 MG cells wererespectively seeded at a density of 2 × 104 cells/well in 24-well plates (Corning-Coaster, Tokyo, Japan), incubated for 72h, and checked under the microscope for similar confluency andmorphology. After this, BCECs and U-87 MG cells wereincubated with DGLs-PEG/DNA, DGLs-PEG-Y/DNA, orDGLs-PEG-E/DNA NPs loading EMA labeled DNA at theconcentration of 30 μg/well measured by DGLs in the DMEMfor 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-wellplates (Corning-Coaster, Tokyo, Japan), incubated for similarconfluency and morphology. The cells were incubated withthree BODIPY-labeled NPs for 60 min. The cells were washedthree times with phosphate buffer solution (PBS, pH 7.4),trypsinized, and centrifuged at 1200 rpm for 5 min to obtain acell pellet, which was subsequently resuspended in PBS (pH7.4) and analyzed using a flow cytometer (BD, USA). Thefluorescence of BODIPY was collected at 520 nm (FITC-channel). For each sample, 10,000 events were collected andanalyzed. Cells cultured under the normal conditions served asthe control.2.7. Qualitative and Quantitative Evaluation of DGLs

Derivatives/DNA NPs Gene Transfection by U-87 MGCells. 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% confluenceprior to transfection. Before transfection, the medium wasexchanged with fresh serum-free medium. The cells weretreated with different NPs solutions containing 5 μg of plasmidEMA labeled DNA for 4 h at 37 °C. After exchange with a freshserum-containing medium, cells were further incubated for 2days after transfection. Five micrograms of plasmid DNA mixedwith Lipofectamine2000 according to the standard protocol asdescribed in instructions served as positive control. In the caseof qualitative evaluation, the red fluorescence images weretaken using a fluorescence microscope. For luciferase activityassay, medium was removed and the cells were rinsed with PBS(without calcium) and shaken for 30 min at room temperaturein 150 μL of luciferase cell culture lysis reagent supplied by thePromega Luciferase Assay Kit. The lysis solution wascentrifuged at 14000g for 2 min at 4 °C. Luciferase activity in

the supernatant was quantified by a Luciferase Assay System(Promega, Madison, WI, USA), and total amount of cellularprotein was determined by Bradford assay, respectively. Thefinal light unit data of each sample was calculated by the lightunit of each sample measured minus the light unit of blanksample. The results were expressed as light units/mg protein.

2.8. Binding Site Comparison of Two LamininReceptor Binding Peptide and the Cellular UptakeInhibition Assay of the Two Targeted NPs. The twopeptides were labeled with BODIPY and Cy7 (FanboBiochemicals, China) through a bifunctional linker, PEG(MAL-PEG-NHS, MW 2000), with the same ratio ofmodification, respectively. After that, the fluorescent peptideswere purified by ultrafiltration. Then the BCECs grown onsterile glass coverslips were incubated with free Y peptide, Epeptide, and Y plus E peptides before the addition of a mixturesolution of BODIPY labeled YIGSR peptide and Cy7 labeledEPRNEEK peptide (1:1, mol/mol). The binding processproceeded at 4 °C for 2 h. The cells were fixed with 4%paraformaldehyde in PBS for 10 min at room temperature andstained with 300 nM DAPI for 10 min at room temperature.After being washed twice with PBS (pH 7.4), coverslips weremounted and observed by a confocal microscope (Leica TCSSP5, Germany). The emission wavelengths were 488 and 633nm 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 freepeptides before the addition of three NPs at 37 °C, respectively.The uptake inhibition time lasted for 1 h, and cells were washedwith PBS and examined under a fluorescence microscope.

2.9. Tumor Implantation. All animal experiments werecarried out in accordance with guidelines evaluated andapproved by the ethics committee of Fudan University,Shanghai, China. Glioma-bearing mice were prepared byintracranial injection (striatum, 1.8 mm right lateral to thebregma and 3 mm of depth) of 1 × 105 U-87 MG cellssuspended in serum-free media into male nude mice with bodyweight of 20−25 g. At the 18th day, the male nude mice wereintraperitoneally administered with fluorescein potassium andimaged via Xenogen IVIS Lumina System coupled with LivingImage software (Xenogen, Corp, Alameda, CA). The luciferaseintensity was above 3000, which showed a successful tumorformation.

2.10. In Vivo Imaging Analysis. Nude mice wereanesthetized by intraperitoneal injection of 10% chloral hydrate.U-87 MG cells (1 × 105 in 4 μL of PBS 7.4) were implantedinto the right striatum (1.8 mm right lateral to the bregma and3 mm of depth) of the mice by using a stereotactic fixationdevice with mouse adaptor.The three NPs loading EMA-labeled DNA were injected

through the tail vein of tumor-bearing nude mice at a dose of50 μg of DNA/mouse which was fixed based on our previousresearch. Images were taken by CRI in vivo imaging system(CRI, Woburn, MA, USA) 90 min after injection after micewere anesthetized. Then, the mice were sacrificed by injectionof chloral hydrate via tail vein. The principal organs (includingbrain, heart, kidney, liver, lung, and spleen) were removed. Theex vivo distribution of NPs was compared by CRI in vivoimaging system.

2.11. Qualitative Distribution of Gene Expression inMouse Brain and Glioma. The DGLs-PEG-Y/DNA, DGLs-PEG-E/DNA NPs (6:1, DGLs to DNA, w/w, red fluorescenceprotein (RFP) plasmid DNA used in this experiment), and

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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 encapsulationand 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-PEG-E/DNA NPs. The stability of NPs loading DNA against enzyme digestion. Plasmid DNA was released from the NPs by the addition of sodiumheparin separated by agarose gel electrophoresis after enzymes incubation. Lane 6: naked plasmid DNA treated with enzymes. Lanes 7−9: DGLs-PEG/DNA, DGLs- PEG-Y/DNA, and DGLs-PEG-E/DNA NPs with treatment of heparin after enzyme incubation.

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DGLs-PEG/DNA NPs were injected through the tail vein oftumor-bearing mice at a dose of 50 μg of DNA/mouse. About48 h later, animals were anesthetized with 10% chloral hydrateand perfused transcardially with saline followed by 4%paraformaldehyde in PBS. The brains were rapidly removedand postfixed for 24 h, then transferred to PBS containing 30%sucrose at 4 °C until subsidence. Coronal brain sections weremade at a thickness of 30 μm with a cryotome Cryostat (Leica,CM 1900, Wetzlar, Germany) and stained with 300 nM DAPIfor 10 min at room temperature. After washing twice with PBS(pH 7.4), the sections were immediately observed under thefluorescence microscope.2.12. Quantitative Expression of Reporter Gene in

Vivo. The three NPs (6:1, DGLs to DNA, w/w, pGL-3 controlplasmid used in this experiment) were injected into the tail veinof tumor-bearing mice at a dose of 50 μg of DNA/mouse. At 48h after injection, the mice were sacrificed by injection of chloralhydrate via tail vein and the principal organs (including brain,heart, liver, lung, and kidney) were extirpated. The organs werecarefully washed with distilled water and homogenized in 1 mLof lysis reagent (Promega, Madison, WI, USA) using a JY92-IIN tissue homogenizer (Scientz, China). The homogenate wascentrifuged at 14000g for 20 min at 4 °C. Luciferase activity andtotal proteins in the supernatant were measured similarly to thecellular experiment as previously described.2.13. In Vivo Pharmacodynamic Evaluation and

Survival Monitoring. The glioma-bearing mice wererandomized to four groups (10 mice/group). At the 12th,15th, and 18th days after the implantation, each group of micewas treated with intravenous administration of saline, DGLs-PEG/survivin NPs, DGLs-PEG-E/scramble NPs, and DGL-PEG-E/survivin NPs at a dose of 50 μg of DNA/mouse. At the21st day, the mice were monitored through Xenogen IVISLumia 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 atthe 12th, 15th, and 18th days after the implantation aspreviously. At the 21st day, the mice were sacrificed and theglioma tissue was lysed using lysis buffer RIPA at aconcentration of 10 μL/mg. A total of 50 μg of proteinswere resolved on 12% polyacrylamide−SDS gels and thentransferred to PVDF membranes. The membranes wereblocked with 5% nonfat milk in Tris-buffer saline for 1 h, andincubated overnight with primary antibodies for survivin andGAPDH. After three washes, the membranes were incubatedwith anti-rabbit or anti-mouse secondary antibodies conjugatedwith horseradish-peroxidase for 1 h. The levels of specificproteins in each lysate were detected by enhanced chem-iluminescence using ECL plus followed by autoradiography.2.15. RT-PCR for Evaluating Survivin mRNA. The

glioma-bearing mice were randomized to four groups (3mice/group) and treated at the 12th, 15th, and 18th days afterthe implantation as previously. At the 21st day, the mice weresacrificed and the glioma tissues were excised for extraction oftotal RNA. In vivo expression of survivin mRNA was detectedby RT-PCR. Total RNA was extracted by using TRIzol reagent,and possible DNA contamination was removed by digesting theextracted RNA with DNase I. The RNA was purified againusing TRIzol reagent and subjected to the synthesis of first-strand cDNA using a reverse transcription kit. GADPH wasamplified as an internal control. The sequence of the survivin

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 usingStudent’s t test and analysis of variance (ANOVA).

3. RESULTS AND DISCUSSION

The delivery of an exogenous therapeutic gene into the gliomaby systemic administration becomes difficult because of theBBB. Considering the overexpression of laminin receptors inglioma 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. Fluorescentimages of BCECs uptake after the incubation of (A) DGLs-PEG/DNA, (B) DGLs-PEG-Y/DNA, and (C) DGLs-PEG-E/DNA NPsloading EMA-labeled DNA for 1 h. Original magnification: ×200. (D)Comparison of mean fluorescent intensities in BCECs treated withBODIPY-labeled DGLs-PEG/DNA, DGLs-PEG-Y/DNA, and DGLs-PEG-E/DNA NPs for 1 h. Fluorescent images of U-87 MG cellsuptake after the incubation of (E) DGLs-PEG/DNA, (F) DGLs-PEG-Y/DNA, and (G) DGLs-PEG-E/DNA NPs loading EMA-labeledDNA for 1 h. Original magnification: ×200. (H) Comparison of meanfluorescent intensities in U87 MG cells treated with BODIPY-labeledDGLs-PEG/DNA, DGLs-PEG-Y/DNA, and DGLs-PEG-E/DNA NPsfor 1 h. **, p < 0.005; ***, p < 0.001, significance representscomparison between two groups.

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binding peptide can have dual-targeted effects on the BBB andglioma cells in early stage glioma and initiate promptintervention for brain tumor. EPRNEEK derived from thepneumococcal CbpA domain had a key function in guidingStreptococcus pneumonia across the BBB, causing meningitis.22

Herein, constructing EPRNEEK modified DGL-PEG astemplates for gene delivery may overcome the hurdle of theBBB and achieve a desired therapeutic effect of glial tumors.Another YIGSR peptide derived from endogenous substanceswas used in this study for comparison because both peptidesbind to laminin receptors in the BBB.In the therapeutic study, we used a specially designed DNA

cassette survivin, which could interact with cellular transcrip-tional factors in the nucleus and activate transcription ofshRNA genes after transport into tumor cells. Then the shRNAcould be processed into double-stranded siRNAs for silencingsurvivin protein and achieving ideal antitumor efficacy.3.1. Characterization of DGLs-PEG/DNA and Two

DGLs-PEG-peptide/DNA Nanoparticles. The NMR spectralresults (Figure 2A,B) proved the existence of the conjugatestructures of DGLs-PEG-peptide vectors. Meanwhile Ellman’s

reagent was used to determine the percentage of unreactedthiol. And little thiol was detected (data not shown) whichcould be explained as the thiol at the end of peptides reactedwith the MAL at one end of PEG specifically. The three DGLsderivatives/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 shownin Figure 2C, the sizes of the three NPs were all less than 120nm measured by dynamic light scattering (DLS). The twopeptide-modified NPs were slightly larger than the unmodifiedones, which may be explained as the peptide modificationmight increase steric hindrance when DGLs derivatives werecomplexed with plasmid DNA. The TEM results (Figure 2D,E)confirmed that the peptide-modified NPs were spherical andhomogeneous particles with a diameter of 90 nm. It wasreasonable that the size measured by DLS was larger than thatmeasured by TEM because the DLS analysis indicated thehydrated value of the particle size. The size below 120 nm wasthought to be suitable enough for BBB-targeted drug deliverysystems as the size of brain capillary was smaller than that ofother capillaries.The zeta-potential results showed that all three NPs were

positively charged with a surface charge of 3 mV. The positivecharge was mainly attributed to the protonated amino group atthe surface of DGLs. This result also proved that DGLs couldeffectively encapsulate the plasmid to neutralize the negativecharge of DNA. The positive charge may favor the binding withcell membrane, which could facilitate the adsorptive-mediatedendocytosis.Figure 2F showed the electrophoresis results of DGLs

derivatives/DNA NPs. All three NPs with DGLs to DNA atweight ratio of 6:1 could encapsulate DNA completely with noelectrophoresis shift (Figure 2F lanes 3−5) compared withnaked plasmid DNA (Figure 2F lane 2). After treatment withtwo restriction enzymes which could recognize two specificsequences in the plasmid yielding the DNA dividing into twofragments, all three NPs were incubated with heparin which wasnegatively charged and could bind to DGLs, resulting in theDNA releasing from the NPs. The integrity maintained in allthree groups compared with naked plasmid DNA. This resultdemonstrated that the DGLs derivatives could play their DNAprotection effect before uptake of NPs by cells and the stabilityof the NPs was enough to resist the enzymes in surroundingcircumstances.

3.2. Cellular Uptake of the NPs by BCECs and U-87MG Cells. BCECs were incubated with all three NPs for 60min. Fluorescent intensity was compared by both observationunder the microscope (Figure 3A−C) and the flow cytometrymeasurement (Figure 3D). In the case of fluorescent imaging,the signal of cells treated with DGLs-PEG-E/DNA NPs loadingEMA-labeled DNA was the highest, while the cellular uptake ofDGLs-PEG-Y/DNA NPs was less but still higher than that ofDGLs-PEG/DNA NPs. Flow cytometry analysis provided thequantitative result which could verify the results observed influorescent images. Among the BODIPY-labeled DGLsderivatives/DNA NPs, the fluorescent intensity of DGLs-PEG-E/DNA NPs was still the highest. Both qualitativeevaluations observed by microscope and quantitative evalua-tions by flow cytometry analysis were consisted with theprevious 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 weretaken 48 h post-transfection with (A) DGLs- PEG/DNA, (B) DGLs-PEG-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 andexpressed as light units per mg protein. Data represent the mean ±SEM (n = 4). **, p < 0.005, compared between two groups.

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were not affected by serum. These results indicated that theNPs 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. Theresults were shown qualitatively using fluorescent images(Figure 3E−G). The fluorescence intensity exhibited by U-87MG cells had increased when cells were treated with the DGLs-PEG-Y/DNA and DGLs-PEG-E/DNA NPs loading EMA-labeled DNA compared with DGLs-PEG/DNA NPs. Thequantitative result presented by flow cytometry analysisconfirmed the qualitative fluorescence results (Figure 3H).The BODIPY-labeled DGLs were used in this experiment. Themean fluorescent intensities of cells incubated with DGLs-PEG-E/DNA NPs were significant higher than that incubated withDGLs-PEG-Y/DNA and DGLs-PEG/DNA NPs.3.3. In Vitro Gene Transfection. The transfection

efficiency mediated by DGLs series vector/DNA NPs wasassessed in U-87 MG cells. Figure 4A−D gave a qualitativecomparison of the DGLs-PEG/DNA, DGLs-PEG-Y/DNA, andDGLs-PEG-E/DNA NPs as well as positive control lipofect-amine2000/DNA. The RFP expression of DGLs-PEG-Y/DNA,DGLs-PEG-E/DNA NPs and lipofectamine2000, the positivecontrol, 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 alsohigher 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 byEPRNEEK and YISGR, we hypothesized that the binding sitesof the two active-targeting peptides are the cause of that. Toreveal the cellular uptake difference of the two peptide-modifiedNPs, the binding sites of the two peptides with laminin receptorwere compared. When the BCECs were incubated with amixture of the two peptides at the mole ratio of 1 to 1, thecolocation was observed as the yellow spots were overlaysignals from green fluorescence labeled YISGR peptide and redfluorescence labeled EPRNEEK peptide (Figure 5A,E). Thisfigure also revealed that EPRNEEK peptide could bind more tocell membrane compared with YISGR peptide which indicatedthat EPRNEEK peptide might have a higher binding affinity to

laminin receptor. The two peptides were both found to bind tothe membrane. With the addition of free YISGR peptide, thered signal remained at the same level as for untreated cells,while the green signals could not be detected any more (Figure5B,F). As shown in Figure 5C,G, only the YISGR peptide wasobserved to bind to the cell membrane. This result illustratedthat free YISGR peptide could not affect the binding betweenEPRNEEK peptide and laminin receptor. The binding ofYISGR peptide with laminin receptor could not be inhibited byfree EPRNEEK peptide. Furthermore, the binding was notobserved when BCECs were treated with both free YISGRpeptide and EPRNEEK peptide (Figure 5D,H). The resultsdemonstrated that the two peptides had different binding siteson laminin receptor which could not be interfered with by eachother. However, EPRNEEK peptide showed a better affinitythan YISGR peptide, which resulted in the better cellularuptake 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 antitumorefficacy.

3.5. In Vivo Distribution of NPs. The tumor-bearing nudemice were injected with the nontargeted DGLs-PEG/DNA ortwo DGLs-PEG-peptide/DNA NPs loading a fluorescent probeEMA-labeled DNA, respectively. In vivo fluorescent imageswere taken at 90 min after injection. As shown in Figure 6A,EMA-labeled DNA was obviously accumulated in brain in thetumor-bearing mice treated with the DGLs-PEG-E/DNA NPs,while the fluorescence in the brain of the DGLs-PEG-Y/DNANPs treated nude mice was less. This difference is partiallyattributed to the different binding sites of EPRNEEK andYISGR. DGLs-PEG-Y/DNA NPs might be interrupted byendogenous laminins, while DGLs-PEG-E/DNA NPs is lesslikely to be interrupted. In the case of DGLs-PEG/DNA NPs,the fluorescent signal was not so significant, because withoutthe help of active-targeting ligand, NPs cannot penetrate theBBB 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 observedin heart, liver, lung, and kidney. The accumulation in liver

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 labeledYIGSR peptide and Cy7 labeled EPRNEEK peptide (1:1, mol/mol) at 4 °C. Green: BODIPY labeled YIGSR peptide. Red: Cy7 labeled EPRNEEKpeptide. Blue: DAPI labeled cell nuclei. (A−D) Merged images of green signal and red signal. (E−H) Merged images of three fluorescent signals andbright field.

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decreased in mice treated with DGLs-PEG-Y/DNA and DGLs-PEG-E/DNA NPs compared to that treated with DGLs-PEG/DNA NPs, because with PEGylation modification, nano-particles can present a prolonged blood circulation and reducedcapture by the reticuloendothelial system23−25 and also increasethe possibility of binding between active-targeting ligands andtheir receptors in the BBB, which contributes to the brain andglioma site uptake. The DGLs-PEG-Y/DNA NPs wereobserved to possess the most retention in lungs. This resultwas in accordance with previous study findings on the YIGSRpeptide, which has been widely applied in lung-targeted drugdelivery systems.26 The results also showed that the three NPswere found in the kidneys (Figure 6B), suggesting that thekidney is a principal pathway responsible for NP clearance. Thefluorescent signals were more obvious in the tumor site of theone treated with DGLs-PEG-E/DNA NPs (Figure 6C,D). All

Figure 6. In vivo distribution of DGLs derivatives/DNA NPs. (A) Invivo fluorescence images of animals at 90 min after intravenousinjection of DGLs-PEG/DNA, DGLs-PEG-Y/DNA, and DGLs-PEG-E/DNA NPs (from left to right). (B) Ex vivo fluorescence images oforgans harvested 90 min after DGLs-PEG/DNA, DGLs-PEG-Y/DNA,and DGLs- PEG-E/DNA NPs injection (from top to bottom) whereB, 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.

Figure 7. Distribution of RFP gene expression in brains of tumor-bearing 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/DNANPs (C, F, I, and L) 48 h after iv administration. Frozen sections (30μm thick) of caudate putamen (A−C), hippocampus (D−F), corticallayer (G−I), and tumor site (J−L) were examined by fluorescentmicroscopy. The sections were stained with 300 nM DAPI for 10 minat room temperature. Red: RFP. Blue: DAPI stained cell nuclei.Original magnification: ×200.

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

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of the results indicated that DGLs-PEG-E/DNA NPs possessedgood tumor target ability and internalization efficiency.3.6. Qualitative Analysis of Distribution of Gene

Expression in Brain and Tumor Site. RFP expression inthe different parenchyma areas including cortical layer,hippocampus, caudate putamen, and the tumor site at 48 hafter administrated DGLs-PEG/DNA, DGLs-PEG-Y/DNA, orDGLs-PEG-E/DNA NPs are shown in Figure 7A−L. The geneexpression in the four regions treated with DGLs-PEG/DNANPs was less than that treated with peptide-modified NPs. Forthe DGLs-PEG-E/DNA NPs, gene expression observed in thetumor site (Figure 7L) and hippocampus (Figure 7F) washigher than that of the DGLs-PEG-Y/DNA NPs, especially.This is also partially because of the different binding affinities ofEPRNEEK and YISGR.

3.7. Quantitative Analysis of Gene Expression in Vivo.The transfection efficiencies of DGLs-PEG/DNA, DGLs-PEG-Y/DNA, and DGLs-PEG-E/DNA NPs loading pGL-3 controlplasmid in principal organs including the tumor were measuredafter 48 h (Figure 8). The luciferase activity of the DGLs-PEG-E/DNA NPs in the brain was over 1-fold higher than that ofthe DGLs-PEG/DNA NPs. Meanwhile, the luciferase activity ofbrain tissues treated with DGLs-PEG-Y/DNA was less thanthat of brain tissues treated with DGLs-PEG-E/DNA NPs andhigher compared with DGLs-PEG/DNA NPs (Figure 8A). Theluciferase activity of the tumor from DGLs-PEG-E/DNA NPstreated mice was the most among all three groups, which wasthe 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

Figure 9. Antitumor efficacy. (A) Relative enhancement of luciferase signals in luci-U87 glioma-bearing mice after treatment (n = 6). (B) In vivoinhibition of endogenous survivin mRNA expression by RT-PCR. (C) Western blot analysis. In vivo inhibition of survivin protein expression aftertreatment. (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 asan internal control. **, p < 0.01; ***, p < 0.001, significance represents comparison between two groups. (D) Overall survival of glioma-bearingmice (n = 10).

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DGLs-PEG/DNA NPs, whereas heart levels were not changedmarkedly (Figure 8B). The luciferase activity in lung withDGLs-PEG-Y/DNA NPs injection was the highest, whichconfirmed the lung-targeted characteristic of YIGSR peptideapplied in several studies. Considering the potential active-targeting efficiency of EPRNEEK, we chose EPRNEEK peptideas BBB−glioma dual targeting ligand for the following in vivopharmacodynamics evaluation.3.8. Antitumor Efficacy. The clinically therapeutic benefits

are mainly determined based on the quality of life andprolonged survival time of cancer patients.27 Considering thebetter BBB−glioma dual targeting efficiency of EPRNEEK, weused DGLs-PEG-E vehicles as active-targeting gene deliverytemplates. Saline was used as control group and a scrambleDNA was used as control DNA with no therapeutic effect.Survivin-cassette was used as antitumor gene drug for thetreatment. The relative enhancement of luciferase signal intensewas measured, which represented the growth of tumor. After 3times’ treatment, the DGLs-PEG-E/survivin group showed asignificant glioma curing effect with an inhibited luciferasesignal comparing to that of saline, DGLs-PEG/survivin, andDGLs-PEG-E/scramble NPs, which represented a reducedtumor growth (Figure 9A). To further evaluate the antitumorefficacy, the overall survival of the glioma-bearing mice wasestimated (Figure 9D). The control group (saline) exhibited anearly death as a function of time, while DGLs-PEG-E/survivingroups showed a prolonged survival time (Figure 9D). This ismainly attributed to the dual targeting effect of DGLs-PEG-E/survivin NPs and the survivin-cassette. After the internalizationof DGLs-PEG-E/survivin NPs, survivin-cassette could bereleased into cytoplasm and enter into the cell nucleus. TheU6 promoter of the survivin-cassette can interact with cellulartranscriptional factors and then activate transcription of shRNAgenes, which can be processed into double-stranded siRNA fortargeted gene silencing of survivin, which is an anti-cell deathgene that confers resistance of cancer cells to therapeuticagents.28,29

RT-PCR analysis was performed to evaluate the level ofsurvivin mRNA (Figure 9B). The survivin mRNA of in vivoglioma was remarkably inhibited in the group of DGLs-PEG-E/survivin NPs because of the successful internalization of DGLs-PEG-E/survivin NPs and the transcription of survivin-siRNAfrom survivin-cassette.The Western blot analysis also verified the efficient delivery

of DNA-cassettes and the pharmacological effects of survivin-cassette. The level of survivin proteins was markedly down-regulated by the DGLs-PEG-E/survivin NPs, while the survivinproteins remained almost the same in saline, DGLs-PEG/survivin NPs, and DGLs-PEG-E/scramble NPs groups (Figure9C).Therefore, DGLs-PEG-E/survivin NPs showed a great

tumor-targeting ability and an ideal potential for antitumortherapy in vivo pharmacodynamic evaluation.

4. CONCLUSIONSIn this study, two peptides (YIGSR and EPRNEEK) derivedfrom laminin and pneumococcal CbpA, respectively, were usedto modify PEGylated DGLs yielding the laminin receptor-targeted gene vector in the BBB and the glioma. The datacollected in this study indicated that the EPRNEEK peptide-modified NPs were not only more preferable for uptake by bothBCECs and U-87 MG cells but also accumulated in the gliomamore efficiently than the YIGSR peptide-modified NPs.

Binding site analysis revealed that the difference between thetwo peptide-modified NPs was attributed to their differentbinding sites on the laminin receptor. A significant increase ingene expression was observed both in vitro and in vivo, with theadministration of targeted NPs. In vivo pharmacodynamicsevaluation exhibited a prolonged survival time in DGLs-PEG-E/survivin NPs, which revealed that the capability of the active-targeting nanoparticle carrier for simultaneous delivery oftherapeutic agents (survivin-cassette) might enhance theeffectiveness of glioma therapy. In summary, our studysuggested that the EPRNEEK peptide derived from pneumo-coccal CbpA provided an effective modification for DGL-basedDNA-loaded NPs to improve brain glioma dual-targeted genedelivery.

■ AUTHOR INFORMATION

Corresponding Author*Tel: +86-21-5198-0079. Fax: +86-21-5198-0079. E-mail:jiangchen@shmu.edu.cn.

Author Contributions‡Y.L. and X.H. contributed equally to this manuscript.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This work was supported by a grant from National BasicResearch 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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