Enhanced Antiglioblastoma Efficacy of Neovasculature and Glioma

Article pubs.acs.org/molecularpharmaceutics

Enhanced Antiglioblastoma Efficacy of Neovasculature and Glioma Cells Dual Targeted Nanoparticles Lingyan Lv,†,‡,# Yan Jiang,†,# Xin Liu,† Baoyan Wang,† Wei Lv,† Yue Zhao,† Huihui Shi,† Quanyin Hu,§,∥ Hongliang Xin,*,† Qunwei Xu,*,† and Zhen Gu§,∥,⊥ †

Department of Pharmaceutics, School of Pharmacy, Nanjing Medical University, Nanjing 211166, China Department of Pharmacy, Zhangjiagang Hospital of Traditional Chinese Medicine, Affiliated Nanjing University of Chinese Medicine, Zhangjiagang 215600, China § Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United States ∥ Division of Molecular Pharmaceutics and Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States ⊥ Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States ‡

S Supporting Information *

ABSTRACT: Combining treatment of anticancer cells and antiangiogenesis is considered to be a potential targeted strategy for brain glioblastoma therapy. In this study, by utilizing the overexpression of Interleukin 13 receptor α2 (IL-13Rα2) on the glioma cells and heparan sulfate on neovascular endothelial cells, we developed a paclitaxel (PTX) loaded Pep-1 and CGKRK peptide-modified PEG− PLGA nanoparticle (PC-NP-PTX) for glioma cells and neovasculature dual-targeted chemotherapy to enhance the antiglioma efficacy. There were significant differences both on the enhancement of cellular uptake in HUVEC and C6 cells and on the improvement of in vitro antiglioma activity in the respect of proliferation, tumor spheroid growth, tube formation, and migration between PC-NP-PTX and Taxol and NPPTX. As for C6 cells, the IC50 were 3.59 ± 0.056, 2.37 ± 0.044, 1.38 ± 0.028, 1.82 ± 0.035, and 1.00 ± 0.016 μg/mL of Taxol, NP-PTX, PepNP-PTX, CGKRK-NP-PTX, and PC-NP-PTX, and for HUVEC cells, the IC50 were 0.44 ± 0.006, 0.33 ± 0.005, 0.25 ± 0.005, 0.19 ± 0.004, and 0.16 ± 0.004 μg/mL of Taxol, NP-PTX, Pep-NP-PTX, CGKRK-NP-PTX, and PC-NP-PTX, respectively. In vivo distribution assays confirmed that PC-NP-PTX targeted and accumulated effectively at glioma site. PC-NP-PTX showed a longer median survival time of 61 days when compared with Taxol (22 days), NP-PTX (24 days), Pep-NP-PTX (32 days), and CGKRK-NP-PTX (34 days). The in vivo antiglioma efficacy and safety evaluation showed PC-NP-PTX significantly enhanced the antiglioma efficacy and displayed negligible acute toxicity. KEYWORDS: glioma, antineovasculature, dual-targeted nanoparticle, drug delivery, paclitaxel

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INTRODUCTION Glioblastoma (GBM) is one of the leading threats for human health, with only approximately 12% of patients surviving 5 years. Traditional therapy includes surgery, radiotherapy, and chemotherapy, but the result is very poor because of the complex microenvironment.1 As a result of nontargeted capability of chemotherapy agents and several physiologic barriers including blood−brain barrier (BBB) and blood− tumor barrier (BTB), the delivery efficiency of chemotherapy agents to glioma site is greatly limited.2 Traditional biologic strategy for glioma usually concentrates on targeting tumor cells to eradicate them.3−5 However, therapy strategy is not very ideal owing to the gene polymorphism and variability of the tumor cells.6−9 Tumor cells and neovasculature combining chemotherapy as a more © XXXX American Chemical Society

potential therapeutic strategy might offer an ideal option because it can not only kill tumor cells directly, but also destroy the tumor neovasculature, cutting off the supply of nutrition and starving the tumor cells,10,11 holding great potential in reducing the unwanted side effects compared to targeting tumor cells therapy only. In recent years, nanoparticulate drug delivery systems (DDS) have attracted increasing attentions among the various DDS to improve the efficiency of drug delivery to glioma.12,13 Nanoparticles take on a sustained release property and high Received: June 10, 2016 Revised: August 24, 2016 Accepted: September 7, 2016

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DOI: 10.1021/acs.molpharmaceut.6b00523 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Figure 1. Schematic of PTX loaded Pep-1 and CGKRK conjugated dual synergetic targeted nanocarrier for brain glioma treatment via IL-13Rα2 and heparan sulfate mediated endocytosis.

cancers, head, and neck tumors.32−36 However, the clinical use of PTX is greatly hampered by the hydrophobicity and serious side effects.37 Exception of direct toxicity to tumor cells, PTX exhibits inhibition on proliferation, migration, and tube formation of endothelial cell at a very low concentration, which is presenting an attractive antiangiogenic activity.38−40 In this study, we developed a PTX loaded synergetic targeted polymer nanoparticles based on Pep-1 and CGKRK modification (PC-NP-PTX) for glioma treatment, which aimed at improving the antiglioma efficacy via antiangiogenesis and cancerous cytotoxicity (Figure 1). Cellular uptake of the functionalized nanoparticles and antiproliferation assay were investigated in both C6 and HUVEC cells. In vitro tube formation assay and endothelial cell migration assay were performed to confirm the antiangiogenic activity of PC-NPPTX. The in vivo glioma targeting and antiglioma efficacy of PC-NP-PTX were evaluated in intracranial glioma mice model.

drug loading, and most importantly, they have the advantages of passive targeting by the enhanced permeability and retention (EPR) effects of tumor.14 However, there are much higher interstitial pressure and smaller pore size of angiogenesis in glioma compared with peripheral tumors, which prevent the delivery of DDS to the glioma sites.15 Active targeted nanoparticulate DDS, functionalized with tumor-specific ligand, can specifically bind to the receptors overexpressed on glioma vasculature or glioma cells, could improve drug concentration in tumor tissue, and reduce the exposure of normal tissues.16−18 Angiogenesis has been recognized as one of the hallmarks of cancer and plays an important role in tumor growth, evasion, and metastasis.19 Decades ago, the long-standing vision of antiangiogenic therapy had been put forward as an effective antitumor treatment strategy.20 Effective dual-targeted efficacy relies on the specific pathological biomarkers of tumor.21 One well-characterized marker is heparan sulfate, which was overexpressed on neovascular endothelial cells.22 CGKRK (Cys-Gly-Lys-ArgLys) peptide, which is discovered by phage display, possesses high affinity and specificity to heparan sulfate. It was showed that intravenous injected CGKRK could recognize the vessels in tumor but not normal tissues.23 Furthermore, CGKRK could be internalized into the targeted cell cytoplasm and directly bind to mitochondria via heparan sulfate mediated endocytosis.24 Interleukin 13 receptor α2 (IL-13Rα2), one of the subunits of the interleukin-13 receptor, is encoded for a 65 kDa receptor protein. It is overexpressed on established glioma cell lines and primary glioblastoma cell cultures,25−29 which makes IL-13Rα2 an attractive target. Pep-1 (CGEMGWVRC) is a lineal peptide that could bind to IL-13Rα2 with high affinity and specificity, which is capable of crossing the BTB and homing to glioma cells.30 What’s more, Pep-1 conjugated nanoparticles could precisely target the glioma site.14,31 Paclitaxel (PTX) is a widely used chemotherapy drug against various tumors including breast cancers, ovaries tumors, lung

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EXPERIMENTAL SECTION Materials. CGKRK and Pep-1 (CGEMGWVRC) peptides were purchased from GL Biochem Co., Ltd. (Shanghai, China). Methoxyl poly(ethylene glycol)-co-poly(D,L-lactic-co-glycolic acid) copolymer (MePEG-PLGA, 40 kDa) and maleimidylpoly(ethylene glycol)-co-poly(D,L-lactic-coglycolic acid) copolymer (Male-PEG-PLGA, 41.5 kDa) were synthesized by the ring opening polymerization as described before.41 PTX was purchased from Zelang Medical Technology Co., Ltd. (Nanjing, China). Coumarin-6 and DiR (1,1′-dioctadecyl3,3,3′,3′-tetramethyl indotricarbocyanine iodide) were provided by Sigmae-Aldrich (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), BCA kit, and TritonX-100 were purchased from Beyotime Biotechnology Co., Ltd. (Nantong, China). Penicillin−streptomycin, RPMI 1640 medium, fetal bovine serum (FBS), and 0.25% (w/v) trypsin solution were purchased from Gibco BRL B


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Molecular Pharmaceutics (Gaithersberg, MD, USA). All the other solvents were analytical grade. Cell Line. Human umbilical vein endothelial cells (HUVEC) and rat C6 glioma cell lines were obtained from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cell line was cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS), 100 U/ mL penicillin, and 100 μg/mL streptomycin. Cells were cultured in incubators maintained at 37 °C with 5% CO2. All experiments were performed in the logarithmic phase of cell growth. Animals. Balb/c nude mice (male, 4−5 weeks, 20 ± 2 g) were obtained from BK Lab Anima Ltd. (Shanghai, China) and housed at 25 ± 1 °C with free access to food and water. ICR mice (male, 6−7 weeks, 30 ± 2 g) were supplied by Department of Experimental Animals, Nanjing Medical University (Nanjing, China) and maintained under standard housing conditions. All animal experiments were performed in accordance with protocols evaluated and approved by the ethics committee of Nanjing Medical University. Preparation and Characterization of PC-NP. Pep-1 and CGKRK conjugated PEG-PLGA (Pep-PEG-PLGA and CGKRK-PEG-PLGA, respectively) were synthesized and characterized by previously described method. 14 Small molecules were removed by dialysis method and Pep-PEGPLGA and CGKRK-PEG-PLGA were obtained by freezedrying. 1H NMR and FTIR of Pep-PEG-PLGA and CGKRKPEG-PLGA were characterized, respectively. Pep-1 and CGKRK conjugated nanoparticle was prepared with emulsion/solvent evaporation method.14 First, 19 mg of MePEG-PLGA copolymer, 2 mg of Pep-PEG-PLGA, 2 mg of CGKRK-PEG-PLGA, and 1 mg of PTX were dissolved in 1 mL of ethyl acetate, which was then added into 2 mL of 1% (w/v) Poloxamer 188 aqueous solution. The mixture was sonicated by intermittent probe sonication (Xin Zhi Biotechnology 15 Co., Ltd., China) for 5 min at 190 W output in an ice bath. Thereafter, the resulting O/W emulsion was added into 10 mL of 0.5% (w/v) Poloxamer 188 aqueous solution under stirring for 5 min. Ethyl acetate was then evaporated at 40 °C with a vacuum rotary evaporator. Afterward, the resultant bluish solution was filtrated with 0.45 and 0.22 μm filters respectively to remove the aggregates. In order to remove excessive emulsifier, the nanoparticles were concentrated by ultrafiltration and washed twice with ultrapure water. Coumarin-6 or DiR-labeled nanoparticle was prepared with the same procedure as above except 0.2 mg of coumarin-6 or DiR were dissolved in the ethyl acetate solution. The morphology of PC-NP-PTX was observed by transmission electron microscope (TEM) (H-600, Hitachi, Japan). The particle size and zeta potential of the nanoparticles were investigated with dynamic light scattering detector (DLS) (Zs90, Malvern, U.K.). Due to the hydrophobicity of PTX, there was only trace concentration of drug in solution. So, the concentration of free PTX, which was filtered by 0.45 and 0.22 μm in NP-PTX, was so low that it can be negligible.42 In order to determine the encapsulation efficiency (EE) and loading capacity (LC) of the nanoparticles, the nanoparticles were lyophilized by ALPHA 2−4 Freeze-Dryer (0.070 Mbar Vacuum, −80 °C, Martin Christ, Germany) and analyzed by HPLC. The EE% and LC% of the nanoparticles were calculated as indicated below (n = 3).

EE(%) =

amount of PTX in nanoparticles × 100 total amount of PTX added

LC(%) =

amount of PTX in nanoparticles × 100 nanoparticle weight

In vitro PTX release from the nanoparticles was performed under different buffer (pH 7.4 and 5.0) containing 0.5% (w/v) Tween-80. Briefly, 1 mL of NP, Pep-NP, CGKRK-NP, and PCNP loaded with PTX (0.09 mg) were subjected into a dialysis bag (MWCO 7000 Da), which were then sealed and incubated in 40 mL of release medium at 37 °C at a shaking speed of 120 rpm for 72 h. At appropriate time points, 0.4 mL aliquots were taken out and replaced with equal amounts of fresh release medium immediately. Finally, samples were analyzed by HPLC analysis as described previously.17 Cellular Uptake of Coumarin-6 Fluorescence Labeled Nanoparticles in C6 Cells and HUVEC. Qualitative analysis of cellular uptake of coumarin-6-labeled nanoparticles was performed by fluorescent microscopy. C6 cells and HUVEC cells were seeded into a 6-well plate at the density of 3 × 105 cells/well and allowed to attach for 24 h, respectively. Then the cells were exposed to coumarin-6-labeled NP, Pep-NP, CGKRK-NP, and PC-NP as the coumarin-6 concentrations ranged from 25 to 50 ng/mL, respectively. After incubation for 1 h, the cells were washed twice and fixed with 4% formaldehyde for 15 min, and then subjected to fluorescent microscopy analysis (Imager A1, Zeiss, 7 Germany). For quantitative experiment, C6 cells and HUVEC cells were seeded into a 24-well plate at the density of 1 × 105 cells/well. After incubation for 24 h, the media were replaced with nanoparticles at the coumarin-6 concentrations ranged from 200 to 2000 ng/mL at 37 °C for 1 h. In order to study the effects of incubation time on cellular uptake, C6 cells and HUVEC cells were incubated with nanoparticles as the coumarin-6 concentrations were 500 ng/mL for 0.5, 1, 2, and 4 h at 37 °C, respectively. At the end of each time point, the cells were washed with cold PBS, and then lysed by 400 μL of 1% TritonX-100 per well for 10 min. Afterward, an aliquot of the cell lysate from each well was measured using the BCA protein assay. The fluorescence intensity of each well was analyzed by HPLC. For receptor competitive inhibition assay, Pep-1, CGKRK, and PC were added to the wells in advance at a concentration of 800 ng/mL. After incubation at 37 °C for 30 min, the peptides were replaced with media containing the coumarin-6labeled PC-NP, followed by above-mentioned steps. Antiproliferation Assay and Tube Formation Assay. The cytotoxicity of PTX loaded Pep-NP, CGKRK-NP, and PCNP was evaluated in C6 cells and HUVEC cells by MTT assay. Briefly, C6 cells and HUVEC cells were seeded in 96-well plate at the density of 2 × 103 cells per well. After attachment for 24 h, the cells were incubated with Taxol, NP-PTX, Pep-NP-PTX, CGKRK-NP-PTX, and PC-NP-PTX for 48 h at a series of different concentration (from 1 ng/mL to 10 μg/mL), respectively. Thereafter, cells were exposed to 20 μL of MTT (5 mg/mL) in each well for 4 h. Then the medium was replaced with 200 μL of DMSO to solubilize formazan crystals. Cells were incubated at room temperature away from light for another 15 min. The absorbance at 490 nm was detected using a microplate reader (Thermo Multiskan MK3, USA). Cells without exposure to the PTX formulations were used as control. C


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For qualitative study, the intracranial C6 glioma bearing mice were obtained as described above, and after housing for 14 days, coumarin-6-labeled NP, Pep-NP, CGKRK-NP, and PCNP were given to the mice intravenously at the dose of 0.2 mg/ kg coumarin-6, respectively. Two hours later, the mice were anesthetized and perfused with saline and 4% paraformaldehyde, respectively, and the brains were harvested and fixed in 4% paraformaldehyde, dehydrated with 15%, 30% sucrose solution, embedded in OCT (Sakura, Torrance, CA, USA). Each brain was cut into 5 μm by frozen section and subjected to fluorescent microscopy analysis after nuclei stained with DAPI. For quantitative studies, the mice bearing intracranial C6 glioma were divided into five groups randomly, and intravenously injected with Taxol, NP-PTX, Pep-NP-PTX, CGKRKNP-PTX, and PC-NP-PTX at the dose of 8 mg/kg PTX, respectively. At each time point (0.5, 1, and 4 h, n = 5 at each time point) after injection, brain and glioma of the mice were collected. The tissues were homogenized in 0.9% sodium chloride solution with 1% TritonX-100 after the weight measurement. The supernatant was obtained after centrifugation. In order to determine the content of PTX, 100 μL of supernatant of tissue homogenates (or 100 μL plasma) were mixed with 100 μL methanol containing 100 ng/mL docetaxel (internal standard). Thereafter, 1 mL of ether was added to extract the PTX and docetaxel. After drying the organic phase under N2, the residue was dissolved with 200 μL of 80% methanol solution and analyzed by HPLC−MS/MS (Agilent 1200 LC−MS, USA). The antiglioma efficacy of the formulations was also evaluated. ICR mice bearing intracranial C6 glioma were divided into six groups (n = 7 or 8) randomly and treated with saline, Taxol, NP-PTX, Pep-NP-PTX, CGKRK-NP-PTX, and PC-NP-PTX (at an equal dose of 10 mg/kg PTX) via tail vein injection every other day for four times, respectively. The survival time were presented as Kaplan−Meier plots and analyzed with a log-rank test. In Vivo Safety Evaluation. Twenty male ICR mice were divided into four groups (n = 5) randomly, and intravenously injected with blank Pep-NP, CGKRK-NP, and PC-NP (100 mg/kg) or saline per day for six times, and the body weight was monitored each day. After 24 h of the last administration, blood samples and tissues (heart, liver, spleen, lung, kidney, and brain) were collected for hematologic and histochemistry analysis. The serum aspartate transaminase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), and creatinine (CRE) levels were assayed using Hitachi 7080 Chemistry Analyzer (Hitachi Ltd., Japan). The tissues were fixed with paraformaldehyde for 24 h and embedded in paraffin. Each brain was cut into 5 μm by paraffin section, processed for routine hematoxylin and eosin (H&E) staining, and then visualized under fluorescent microscope (Imager A1, Zeiss, Germany). Statistical Analysis. All the data were expressed as means ± SD. Statistical significance among multiple-group was examined using one-way ANOVA. Survival time was analyzed with SPSS 20.0 software. Differences were considered significant when *P < 0.05, **P < 0.01, and *** P < 0.001, respectively.

The cell cytotoxicity of blank nanoparticles was evaluated also by the same method with the concentration ranged from 0.1 to 1000 μg/mL. The tube formation assay and the endothelial cell migration assay were performed to evaluate the in vitro antiangiogenic effect. Matrigel was dissolved at 4 °C overnight, with each well of prechilled 96-well plates coated with 50 μL of Matrigel followed by incubation at 37 °C for 45 min to allow the Matrigel to polymerize. HUVEC cells (5 × 104) were added to each well on the layer of Matrigel, together with various concentration of Taxol, NP-PTX, Pep-NP-PTX, CGKRK-NPPTX, and PC-NP-PTX at different concentrations of PTX (0.85, 4.25, and 8.50 ng/mL). After 8 h of incubating at 37 °C in a 95:5 (v/v) mixture of air and CO2, the endothelial cells were photographed using an inverted microscope. The medium containing no PTX was set as controls. Endothelial Cell Migration Assay. Migration assay of HUVEC was performed in a 24-well Transwell Boyden Chamber (Costar, MA, USA) via a polycarbonate filter with the pore size of 8 μm.43 In brief, 100 μL of cell suspension (2 × 106 cells/mL) treated with a series of different PTX concentration (from 0.85 to 42.5 ng/mL) of Taxol, NP-PTX, Pep-NP-PTX, CGKRK-NP-PTX, and PC-NP-PTX was loaded into the upper compartment of the chamber. A total of 600 μL of 1640 medium was added to the lower compartment. After incubation at 37 °C for 12 h, the chamber was disassembled. Then the membrane was removed, rinsed with PBS, and fixed in 4% formaldehyde for 15 min. The upper surface was scraped to remove the nonmigrant cells, and the migrant cells on the lower surface were stained with 0.1% crystal violet for 25 min and then visualized and photographed using a microscope (Olympus, DP50, Tokyo, Japan). Five random fields from each well were selected, and in total, three wells in each group were examined. Inhibition of Tumor Spheroid Growth. The antigrowth ability on tumor spheroids was evaluated by observing the size of spheroids following different PTX formulations treatment. Briefly, the selected tumor spheroids (2 days incubation) were treated with Taxol, NP-PTX, Pep-NP-PTX, CGKRK-NP-PTX, and PC-NP-PTX at PTX concentration of 5 μg/mL every 3 days for three times. The size of tumor spheroids was carefully observed under an invert microscope (Chongqing Optical and Electrical Instrument, Co., Ltd., Chongqing, China). The spheroids treated with 1640 were used as the negative control. In Vivo Biodistribution and Antiglioma Effect of PCNP. In vivo biodistribution effect of PC-NP was evaluated through the real-time imaging assay of intracranial gliomabearing mice. DiR was utilized as the fluorescent probe to monitor the real-time distribution of nanoparticles. To establish the intracranial glioma bearing mice model, 12 nude mice divided into four groups randomly were injected C6 cells (5 × 105 cells suspended in 5 μL of PBS) into right corpus striatum using a stereotaxic apparatus. Fourteen days later, four groups of glioma bearing mice were intravenously administrated with 200 μL of DiR-labeled NP, Pep-NP, CGKRK-NP, and PC-NP (0.8 mg/kg) via the tail vein, respectively. The concentration of DiR was measured by fluorescent spectrophotometer. The fluorescent images of nude mice were acquired at predetermined time points (4 and 24 h) via an in vivo imaging system (Caliper, USA). After administration for 24 h, all mice were sacrificed and the tumor-bearing brains were harvested for fluorescent imaging.

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RESULTS Characterization of Nanoparticles. The structures of male-PEG-PLGA, Pep-PEG-PLGA, and CGKRK-PEG-PLGA

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Molecular Pharmaceutics Table 1. Characterization of PTX-Loaded NP, Pep-NP, CGKRK-NP, and PC-NP formulation NP Pep-NP CGKRK-NP PC-NP

particle size (nm) 89.3 93.2 95.2 97.3

± ± ± ±

1.2 2.4 2.1 1.4

zeta potential (mV) −32.5 −33.6 −25.4 −30.8

± ± ± ±

polydispersity index (PDI)

1.2 1.3 1.1 1.4

0.162 0.117 0.150 0.146

EE% 84.8 83.2 78.84 81.7

± ± ± ±

LC% 2.3 3.2 2.8 2.7

4.4 3.6 3.4 3.5

± ± ± ±

0.1 0.2 0.1 0.1

Zeta potential of NP formulation was −32.5 ± 1.2 mV, while that of Pep-NP, CGKRK-NP, and PC-NP was −33.6 ± 1.3, −25.4 ± 1.1, and −30.8 ± 1.4 mV (Table 1), respectively. The LC of NP, Pep-NP, CGKRK-NP, and PC-NP were 4.4 ± 0.1%, 3.6 ± 0.2%, 3.4 ± 0.1%, and 3.5 ± 0.1%, respectively, with the EE 84.8 ± 2.3%, 83.2 ± 3.2%, 78.8 ± 2.8% and 81.7 ± 2.7%, respectively (Table 1). The in vitro accumulative release profiles of PTX from different formulations were shown in Figure 2C,D. At the end of 72 h (Figure 2C), the accumulative PTX release from NP was 70.3 ± 0.1%, and that of Pep-NP-PTX, CGKRK-NP-PTX, and PC-NP-PTX was 76.9 ± 5.4%, 77.5 ± 4.5%, and 74.4 ± 3.7% in pH 5.0 PBS, respectively, indicating the sustained release pattern of PTX in cytoplasm. Cellular Uptake in C6 Cells. The cellular uptake characteristic of nanoparticles in C6 cells was investigated qualitatively by fluorescent microscopy, and coumarin-6 was used as the fluorescent tracer probe. From Figure 3A, it was

were determined by 1H NMR spectroscopy. The value 7.26 ppm was attributed to the peak of CDCl3. In the spectrum of male-PEG-PLGA, the methyl protons of PLGA were at 1.5 ppm and the methylene protons of PEG was at 3.6−3.7 ppm (Figure S1A); the peak of maleimide group of the polymer could be discerned at 6.7 ppm before reaction (Figure S1A), which disappeared after reaction with Pep-1 or CGKRK, whereas the PLGA and PEG segment still presented at 1.5 and 3.6−3.7 ppm (Figure S1B,C), suggesting that Pep-1 and CGKRK were conjugated with male-PEG-PLGA copolymer, respectively.14,44 The FTIR spectra of male-PEG-PLG, Pep-PEG-PLGA, and CGKRK-PEG-PLGA were shown in Figure S2. The spectrum of male-PEG-PLGA showed a weak CO stretching vibrational absorption at 1746.82 cm−1 (Figure S2A), which was also present in the spectrum of Pep-PEG-PLGA and CGKRK-PEGPLGA, respectively (Figure S2B,C). At the same time, there was a weak CO stretching vibrational absorption at 1647.69 cm−1, 1657.77 cm−1 and a very broad absorption at 3414.59 cm−1, 3247.61 cm−1 (Figure S2B,C), which were attributed to the N−H stretching vibrational absorption of Pep-1 and CGKRK segment. However, these two characteristic vibrational absorption were absent of male-PEG-PLGA. Collectively, these results demonstrated the successful synthesis of Pep-PEGPLGA and CGKRK-PEG-PLGA. The PC-NP was prepared via emulsion/solvent evaporation. The physical characterizations of PTX-loaded NP, Pep-NP, CGKRK-NP, and PC-NP were shown in Table 1. The mean particle size of NP, Pep-NP, CGKRK-NP, and PC-NP were 89.3 ± 1.2, 93.2 ± 2.4, 95.2 ± 2.1, and 97.3 ± 1.4 nm, respectively. TEM exhibited the spherical shape of PC-NP-PTX (Figure 2A).

Figure 3. Fluorescent images of cellular uptake in C6 cells at the coumarin-6 concentration of 25 and 50 ng/mL at 37 °C for 1 h (A). Original magnification: 200×. Uptake of coumarin-6-loaded NP, PepNP, CGKRK-NP, and PC-NP by C6 cells at concentrations from 200 to 2000 ng/mL for 1 h at 37 °C (B). Uptake of coumarin-6-loaded NP, Pep-NP, CGKRK-NP, and PC-NP by C6 cells at 37 °C for 0.5− 4.0 h at the coumarin-6 concentration of 500 ng/mL (C). Data represented mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 mean significant difference compared with plain NP at 37 °C.

seen that C6 cells treated with coumarin-6-loaded NP, Pep-NP, and PC-NP exhibited fluorescent intensities corresponding to coumarin-6 concentrations. The cellular associated fluorescence intensities of Pep-NP and PC-NP were obviously higher than that of NP at all the detected concentrations. Quantitatively, uptake of coumarin-6-loaded NP, Pep-NP, CGKRK-NP, and PC-NP by C6 cells showed the concen-

Figure 2. TEM image (A) and particle size and size distribution (B) of PC-NP-PTX. PTX release profiles from various nanoparticles in PBS (pH 5.0) (C) and PBS (pH 7.4) (D) with 0.5% (w/v) Tween-80. E


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Quantitatively, the cellular uptake of coumarin-6-loaded NP, Pep-NP, CGKRK-NP, and PC-NP in HUVEC cells showed the concentration-dependent and time-dependent behaviors (Figure 4B,C). Compared with NP, the cellular fluorescence intensities of CGKRK-NP and PC-NP were significantly higher at various different concentrations and all experiment time points. The cellular uptakes of CGKRK-NP and PC-NP were 1.77, 1.50, 1.52, 1.59, 1.51-fold and 2.02, 1.72, 1.73, 1.82, 1.68fold higher on HUVEC cells when compared with that of NP at 37 °C at the coumarin-6 concentration of 200, 400, 800, 1000, and 2000 ng/mL, respectively. In addition, the time-related experiment exhibited that the fluorescence intensities of CGKRK-NP and PC-NP on HUVEC cells were significantly enhanced when compared with that of NP at the incubation time ranging from 0.5−4 h. Meanwhile, the uptake of Pep-NP group was greater than that of the NP group, but still considerably lower than that of the CGKRK-NP and PC-NP group, which suggested that Pep-1 could not bind to the HUVEC cells as effective as CGKRK. Through the qualitative and quantitative analysis, CGKRK modification increased the cellular uptake, which was consistent with the overexpression of heparan sulfate on HUVEC cells. In receptor competitive inhibition assay (Figure S3), in C6 cells, preincubation with free Pep-1 and PC peptides obviously inhibited the cellular uptake of PC-NP, which suggested that the endocytosis of PC-NP by C6 cells was mediated by IL13Rα2. Meanwhile, in HUVEC, preincubation with free CGKRK and PC peptides also blocked the uptake of PC-NP, suggesting that the endocytosis of PC-NP by HUVEC cells was mediated by heparan sulfate. Antiproliferation Assay and Tube Formation Assay. The cell cytotoxicity of blank nanoparticles was seen in Figure S4; the cytotoxicity of Cremophor EL was obviously higher compared with nanoparticles when the incubation concentration was more than 10 μg/mL in both C6 and HUVEC cells. However, blank NP, CGKRK-NP, Pep-NP, and PC-NP did not show obvious cytotoxicity at determined concentration. These results suggested that blank NP, CGKRK-NP, Pep-NP, and PC-NP were not toxic to C6 and HUVEC cells probably due to the biocompatibility of the block polymers. In vitro antiproliferation effect of different PTX formulations on C6 cells and HUVEC cells was evaluated using the MTT assay. The IC50s were 3.59 ± 0.056 μg/mL for Taxol, 2.37 ± 0.044 μg/mL for NP-PTX, 1.38 ± 0.028 μg/mL for Pep-NPPTX, 1.82 ± 0.035 μg/mL for CGKRK-NP-PTX, and 1.00 ± 0.016 μg/mL for PC-NP-PTX (Figure 5A) on C6 cells. Consistently, PC-NP-PTX induced the highest cytotoxicity against C6 cells among these formulations. The IC50s were 0.44 ± 0.006 μg/mL for Taxol, 0.33 ± 0.005 μg/mL for NP-PTX, 0.25 ± 0.005 μg/mL for Pep-NP-PTX, 0.19 ± 0.004 μg/mL for CGKRK-NP-PTX, and 0.16 ± 0.004 μg/mL for PC-NP-PTX (Figure 5B) on HUVEC cells. It was demonstrated that HUVEC cells were the most sensitive to PC-NP-PTX among the examined formulations. Various kinds of cells are involved in the process of angiogenesis, one of the most important steps is tube formation of endothelial cell. We evaluated the effect of different PTX formulations by plating HUVEC on Matrigel to form the functional tubes. As shown in Figure 5C, HUVEC treated with culture medium treatment (blank control) formed extensive and enclosed tube networks (a robust tube-like structure). In contrast, such tube formation ability was restrained following the treatment with Taxol and all the PTX-loaded nanoparticles,

tration-dependent and time-dependent behaviors (Figure 3B,C). Compared with NP, the cellular fluorescence intensities of Pep-NP and PC-NP were significantly higher at various different concentrations and all experiment time points. The cellular uptakes of Pep-NP and PC-NP were 1.27, 1.36, 1.23, 1.25, 1.29-fold and 1.45, 1.58, 1.32, 1.51, 1.35-fold higher on C6 cells in comparison with that of NP at 37 °C at the coumarin-6 concentration of 200, 400, 800, 1000, and 2000 ng/mL, respectively. In addition, the time-related study exhibited that the fluorescence intensities of Pep-NP and PC-NP on C6 cells were significantly enhanced when compared with that of NP at the incubation time ranging from 0.5 to 4 h. At the same time, we found the cellular fluorescence intensity of CGKRK-NP was slightly higher than that of NP at each concentration and time point, we speculated that there was also heparan sulfate overexpression of tumor cells.45 From the qualitative and quantitative investigation, it was seen that the cellular accumulation of Pep-NP and PC-NP was significantly higher than that of the plain NP, suggesting that Pep-1 could specifically bind to IL-13α2 receptor, which was highly expressed of C6, mediating the nanoparticles internalization via active endocytosis. Cellular Uptake in HUVEC. The HUVEC uptake characteristic of nanoparticles was investigated qualitatively by fluorescent microscopy, and coumarin-6 was used as the fluorescent tracer probe. From Figure 4A, HUVEC cells treated

Figure 4. Fluorescent images of cellular uptake in HUVEC at the coumarin-6 concentration of 25 and 50 ng/mL at 37 °C for 1 h (A). Original magnification: 200×. Cellular uptake of coumarin-6-labeled NP, Pep-NP, CGKRK-NP, and PC-NP in HUVEC at concentrations from 200 to 2000 ng/mL for 1 h at 37 °C (B). Uptake of coumarin-6loaded NP, Pep-NP, CGKRK-NP, and PC-NP by C6 cells at 37 °C for 0.5−4.0 h at the coumarin-6 concentration of 500 ng/mL (C). Data represented mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 mean significant difference compared with plain NP at 37 °C.

with coumarin-6-loaded NP, CGKRK-NP, and PC-NP exhibited the fluorescent intensities corresponding to coumarin-6 concentrations. The fluorescence intensity of CGKRK-NP and PC-NP was obviously higher compared with NP group at all the detected concentrations. F


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Figure 5. In vitro antiproliferation effect of different PTX formulations on C6 cells (A) and HUVEC cells (B) at different concentration at 48 h. Data represented mean ± SD (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 denote significant difference compared with NP-PTX. Effect of various PTX formulations including Taxol, NP-PTX, PepNP-PTX, CGKRK-NP-PTX, and PC-NP-PTX on HUVEC tube formation at the PTX concentration from 0.85 to 8.50 ng/mL (C). Original magnification: 100×. Figure 6. Effect of various PTX formulations including Taxol, NPPTX, Pep-NP-PTX, CGKRK-NP-PTX, and PC-NP-PTX on HUVEC migration at the PTX concentration from 0.85 to 42.5 ng/mL. HUVEC cells treated with blank 1640 served as the control. Purple stained cells indicated the number of HUVEC migrated to the lower membrane. Photomicrographs showed HUVEC migration by Transwell test (A). Original magnification: 100×. Cell number/field of HUVEC migration (B). Data represented mean ± SD (n = 3). **P < 0.01, ***P < 0.001 significantly more than that of NP-PTX.

among which PC-NP-PTX most efficiently blocked angiogenesis at drug concentrations of 0.85, 4.25, and 8.50 ng/mL, suggesting a potential for decreasing angiogenesis. Endothelial Cell Migration Assay. The antimigration of different formulations was shown in Figure 6. The majority of HUVEC cells migrated to the other side of the membrane of the control group (Figure 6A). Compared to Taxol, NP-PTX and Pep-NP-PTX exhibited much higher activity of inhibiting migration with fewer cells in each field. CGKRK-NP-PTX and PC-NP-PTX group showed significant higher ability to inhibit the migrating of HUVEC cells the other side compared to NP at PTX concentrations of 0.85, 8.50, and 42.5 ng/mL (Figure 6B). Inhibition of Tumor Spheroid Growth. In the aspect of simulating the internal solid tumor tissue, multicells tumor spheroids are superior to monolayer tumor cells. By taking advantage of viable rim, with gradients of oxygen tension, nutrients, catabolites, and cell proliferation, 3D tumor spheroids are almost the same as internal tumor microenvironment.46 Therefore, multicells 3D glioma spheroids were used in this study to investigate the potential of various PTX formulations to antisolid tumor. The inhibition of tumor spheroids growth was evaluated following the treatment with serum-free 1640, Taxol, NP-PTX, Pep-NP-PTX, CGKRK-NP-PTX, and PC-NP-PTX at the PTX

concentration of 5 μg/mL every 4 days for 2 weeks, respectively. As shown in Figure 7, the tumor spheroids treated with serum-free 1640 kept growing and were tightly organized, and the surface of normal spheroid was smooth. Taxol could decrease the volume, which was significantly smaller than control, but the tumor spheroids were also very compact. The cells on the surface of glioma spheroids treated with NP-PTX were slightly disorganized, and the cell leakage and cell membrane rupture also occurred, suggesting that nanoparticles were more effective than Taxol. Conjugation with Pep-1 or CGKRK could further increase the tumor inhibition effect with more marginal cells loosen from the spheroids. As to the PCNP-PTX group, almost all the cells on the surface were obviously disintegrated and shrunken, losing the three-dimensional structure at day 14 (Figure 7). G


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permeability of vascular changes with the pathological progression of glioma and the presence of BBB/BBTB.47 Pep-NP exhibited a higher concentration in the brain than that of NP, suggesting that Pep-NP could be effectively transported through the BBTB, and targeted the brain glioma due to the overexpression of IL-13α2 on glioma cells. A higher accumulation of CGKRK-NP was seen in glioma site than that of NP, which was mainly contributed by CGKRK-heparan sulfate interaction in vivo. By combining the specific targeting effect of Pep-1 and CGKRK, PC-NP exhibited the highest concentration in the glioma. Furthermore, the images of ex vivo brains also demonstrated that the PC-NP had higher fluorescence intensity at glioma site than that of others at 24 h (Figure 8A). Combining the effect of Pep-1 and CGKRK, PC-NP exhibited the most accumulation in glioma (Figure 8Bd), suggesting tumor cells and glioma neovasculature combining targeted strategy could further enhance the GBM targeting efficiency, which was useful for GBM treatment. In order to quantitatively determine the brain biodistribution of nanoparticles, Taxol, NP-PTX, Pep-NP-PTX, CGKRK-NPPTX, and PC-NP-PTX were given intravenously into intracranial tumor-bearing mice. The amount of PTX in the normal brain tissue and glioma site was determined with HPLC−MS/ MS. There was no significant difference between the five formulations in the normal brain tissue at any time point postinjection (Figure 8C). However, at all-time points, PTX concentrations determined in the glioma section followed the order: PC-NP-PTX > Pep-NP-PTX ≈ CGKRK-NP-PTX > NP-PTX > Taxol (Figure 8C). 0.5, 1, and 4 h after administration, PTX level in the glioma section of PC-NPPTX group were 3.98, 3.38 and 2.29-fold over that of Taxol group, 2.45, 2.51 and 1.97-fold over that of NP-PTX group, 1.24, 1.31 and 1.49-fold over that of Pep-NP-PTX group, and 1.25, 1.56 and 1.39-fold over that of CGKRK-NP-PTX group, respectively, suggesting that more PTX accumulated in the glioma site. The concentrations of PTX in the plasma had no significant difference between the Taxol, NP-PTX, Pep-NPPTX, CGKRK-NP-PTX, and PC-NP-PTX at 0.5, 1, and 4 h (Figure S5), the results excluded the effect of pharmacokinetics on PTX accumulation in glioma. To evaluate the antiglioma effect of the dual-targeting delivery systems, PTX was loaded into different kinds of particles for the treatment of GBM bearing mice (Figure 8D, Table 2). Although Taxol expanded the median survival time (MST) from 17 days to 22 days, no statistical difference was observed between the Taxol group and saline group, which might be explained by the poor glioma targeted ability of Taxol, while NP-PTX significantly prolonged the MST because of the passive targeting effect of nanoparticles. Treated with Pep-NPPTX and CGKRK-NP-PTX significantly prolonged the MST due to the glioma-targeting effect of Pep-1 and CGKRK, which was 1.88 and 2.00-fold higher than that of saline. Dual modification with Pep-1 and CGKRK further improved the treatment outcome because of the combining targeted effect of PC-NP-PTX, which was demonstrated by in vivo imaging. PCNP-PTX group showed a significantly longer MST of 61 days when compared with saline group (17 days, P < 0.001), Taxol group (22 days, P < 0.001), and NP-PTX group (24 days, P < 0.01), which was substantially longer than the Pep-NP-PTX group (32 days) and CGKRK-NP-PTX group (34 days). In Vivo Safety Evaluation. To evaluate the safety of the nanoparticles, we injected saline, blank NP, Pep-NP, CGKRKNP, and PC-NP to healthy mice by the tail vein, and then body

Figure 7. Morphology of 3D glioma spheroids treated with 1640, Taxol, NP-PTX, CGKRK-NP-PTX, Pep-NP-PTX, and PC-NP-PTX on day 0, day 4, and day 8, respectively. Original magnification: 40×.

In Vivo Biodistribution and Antiglioma Effect of PCNP-PTX. In vivo biodistribution of different DiR-labeled nanoparticles were evaluated on C6 glioma bearing mice via noninvasive near-infrared (NIR) imaging. Four hours after administration, the plain nanoparticles were hardly accumulated in the brain, whereas Pep-NP, CGKRK-NP, and PC-NP exhibited an obviously high intensity in the brain. After 24 h, compared with other nanoparticles, NP still exhibited the lowest intensity in the brain site owing to the poor EPR effect (Figure 8A). However, EPR is highly variable as the

Figure 8. In vivo and ex vivo real-time NIR fluorescence imaging of intracranial tumor-bearing mice after treatment of DiR-loaded nanoparticles (A). The distribution of coumarin-6-loaded nanoparticles in glioma sections of intracranial tumor-bearing mice 2 h after treatment (B): NP (a), CGKRK-NP (b), Pep-NP (c) and PC-NP (d); the scale bar was 20 μm. The PTX concentration of Taxol, PTXloaded NP, Pep-NP, CGKRK-NP, and PC-NP in brain and glioma (C) at different times following i.v. administration to intracranial glioma-bearing ICR mice at a single 8 mg/kg dose of PTX (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 significantly lower than that of PCNP-PTX. Kaplan−Meier survival curves of C6 glioma-bearing mice treated with different PTX formulations at a dose of 10 mg/kg PTX on day 2, 4, 6, and 8 post-implantation (D). H


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Table 2. In Vivo Antiglioma Efficacy of Various PTX Formulations Using Intracranial C6 Glioma Mice Model (n = 7 or 8)a groups saline Taxol NP-PTX Pep-NP-PTX CGKRK-NP-PTX PC-NP-PTX a

dose (mg/kg) 10 10 10 10 10

MST (days)

median (days)

compare with saline

compare with taxol

compare with NP

± ± ± ± ± ±

17 22 24 32 34 61

p > 0.05 d c c b

p > 0.05 d d b

d d c

17.3 21.8 24.2 31.9 34.3 60.7

1.2 2.3 3.5 2.2 3.1 4.0

MST: mean survival time. b***P < 0.001. c**P < 0.01. d*P < 0.05 of log-rank analysis.

NP, Pep-NP, CGKRK-NP, and PC-NP showed a similar size distribution and zeta potential, indicating that these nanoparticles might have similar systemic pharmacokinetic behavior (Figure S5). Due to the hydrophobicity of PTX, there was only trace concentration of drug in solution. Therefore, 0.5% (w/v) Tween-80 was added into phosphate buffer solution to reach sink condition in PTX release assay. The results showed that the various formulations presented almost the same release behavior in both pH 7.4 and 5.0, indicating the modification of Pep-1 and CGKRK peptide did not alter the release behavior of PTX. The mechanism of drug release may be the combination of diffusion through the nanoparticles matrix and polymer matrix biodegradation. In our previous study, less than 10% of coumarin-6 released from the nanoparticles within 48 h.14 In PBS (pH 7.4) lower than 1% of DiR was released while in 10% FBS medium there was approximately 3% after 72 h.58 Therefore, the fluorescence signals from DiR or coumarin-6 could reflect the position of nanoparticles. From the cellular uptake of C6 and HUVEC cells, these results exhibited that PC-NP was internalized by both C6 cells and HUVEC cells more efficiently than NP in the concentration- (Figures 3B and 4B) and time-dependent (Figures 3C and 4C) manners. Furthermore, the C6 cellular uptake of PC-NP was obviously blocked by the free Pep-1 and PC peptides, indicating that the endocytosis of PC-NP on C6 cells was mediated by IL-13Rα2. Meanwhile, in HUVEC cellular uptake of PC-NP, it was also obviously inhibited by free CGKRK and PC peptides, which indicated the endocytosis of PC-NP by HUVEC cells was mediated by heparan sulfate. This high specificity was mainly due to the overexpression of IL13α2 in C6 cells and heparan sulfate in HUVEC cells, indicating Pep-1 and CGKRK peptide functionalized nanoparticles mediated cellular endocytosis. Taken together, it could be speculated that PC-NP might show significant dual-targeting efficiency in vivo. The enhanced cellular uptake of nanoparticles could improve the antiproliferative activity of model drug. The antiproliferative effect of PTX nanoparticles was significantly enhanced after conjugating with Pep-1 and CGKRK peptide, which was consistent with the results of cellular uptake of coumarin-6labled PC-NP (Figures 3 and 4), indicating enhanced internalization of nanoparticles after functionalization with Pep-1 and CGKRK peptide led to much higher intracellular PTX concentration in the both cells and thus more efficient anticancer activity. The strong cytotoxicity of PC-NP-PTX on tumor cells might also prevent local invasion, which often occurred during antiangiogenic therapy alone. Moreover, PC-NP-PTX showed stronger tube formation and HUVEC migration than NP-PTX and Taxol. Antiproliferation assay illustrated that PC-NP-PTX significantly increased

weight, serum biochemical analyses, and histopathology were monitored as a marker of the toxicity. During the study period we can see that there were no deaths and serious body weight loss in all groups (Figure S6). H&E staining was used to characterize the toxicity to the organs. The H&E staining for the liver, heart, spleen, kidney, lung, and brain section of PepNP, CGKRK-NP, and PC-NP group showed that there was no evidence of abnormal and inflammatory cell infiltration in sections (Figure S7), indicating Pep-NP, CGKRK-NP, and PCNP did not cause toxicity on organs of the mice.

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DISCUSSION In recent years, antiangiogenic therapy has received more and more attention. As glioblastma is one of the most vascularized tumors, angiogenesis is a key prerequisite for glioma growth, metastasis, and progression.48 However, because of the existence of the resistance to the proapoptotic effect of chemotherapy along with the enhanced metastatic and invasive possibility of glioma cells, the curative effect of antiangiogenic therapy only is disappointing.49−52 Therefore, glioma neovascular and cells combining targeted are urgently needed to prevent tumor angiogenesis and inhibit tumor growth. In this study, neovasculature and glioma cells dual targeted nanoparticles were developed to enhance the antiglioblastoma efficacy. When the PC-NP-PTX was intravenously administrated, some of the nanoparticles can get into tumor site based on EPR effects. When the targeted nanoparticles were internalized by the endothelial cells on the tumor vasculature via heparan sulfate mediated endocytosis, the tumor angiogenesis blood vessels may be destroyed, and the nanoparticles can further target the tumor cells through IL-13Rα2 mediated endocytosis.53 Meanwhile, the targeted nanoparticles with high transcytosis capacity could be considered a material of choice to cross the tumor angiogenesis blood vessels as well as to specifically recognize their receptors (IL-13Rα2) present on the glioma cells for directing the site specific release of anticancer drug.54 First, PTX-loaded PEG-PLGA nanoparticles were constructed via emulsion-evaporation method. Pep-1 and CGKRK peptide were decorated to nanoparticles via maleimide-mediated covalent binding procedure. The size of NP was increased from 89.3 ± 1.2 to 97.3 ± 1.4 nm after modification with Pep-1 and CGKRK peptide, the size of particles was slightly increased but still smaller than 100 nm, which was suitable for tumor drug delivery.55 When the size was below 200 nm, it was beneficial for nanoparticles passive targeting to the tumor site through the EPR effect.56,57 After modification, zeta potential of Pep-NP decreased from −32.5 ± 1.2 to −33.6 ± 1.3 mV, as Pep-1 peptide contained negative charge amino acids, while CGKRK-NP was increased from −32.5 ± 1.2 to −25.4 ± 1.1 mV due to positive charge amino acids (lysine and arginine) of CGKRK peptides. The results of I



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CONCLUSION In this study, we investigated a tumor cells and neovasculature combination targeted strategy of nanocarrier, which was validated to enhance the antiglioma efficacy. The acquired PC-NP was observed spherical in shape with a diameter of 97.34 ± 1.45 nm and a zeta potential of −30.8 ± 1.45 mV. The Pep-IL-13Rα2 and CGKRK-heparan sulfate mediated recognition and internalization significantly facilitated the uptake of PC-NP on C6 and HUVEC cells, thus inducing a strengthened antiproliferation effect with an IC50 of 1.00 ± 0.016 μg/mL and 0.16 ± 0.004 μg/mL, respectively. Furthermore, PC-NP-PTX significantly improved the inhibitory effects of PTX on the growth of tumor spheroids, HUVEC tube formation, and migration assay, which showed significantly stronger antiangiogenic activity than Taxol and NP-PTX. In vivo NIR imaging and tumor distribution showed that PC-NP displayed a much better tumor targeting and higher accumulation within the glioma. More importantly, PC-NP-PTX exhibited the most powerful antitumor activity with the longest survival time in mice bearing intracranial C6 glioma. These findings indicated that Pep-1 and CGKRK-conjugated targeted PEG-PLGA nanoparticles hold great potential for improving the antiangiogenic and antiglioma efficacy of the loaded cargoes.

cytotoxicity in HUVEC cells (Figure 5B), which could be due to the higher cellular accumulation of PTX via the CGKRK peptide-mediated endocytosis. Combined together, these results have confirmed that PC-NP-PTX significantly improved the antiangiogenic ability of PTX. It could be attributed to the high affinity between CGKRK and heparan sulfate, which was specifically overexpressed on the endothelial cells of neovasculature. In vitro antitumor spheroids growth experiment, at day 14, the tumor spheroids of PC-NP-PTX group lost the threedimensional structure, exhibiting much stronger inhibitory effects on tumor spheroids when compared with NP-PTX and Taxol. The tumor spheroid growth inhibition effect of different formulations was consistent with cell uptake and antiproliferation assay, suggesting the greater inhibition effect of PC-NPPTX was owing to the dual targeting delivery system, which could induce the apoptosis of not only the surface cells but also the interior cells of the spheroids. To further investigate the in vivo glioma targeting effect of PC-NP, we performed near-infrared (NIR) imaging study. Distribution of PC-NP in the glioma site was much more than NP group (Figure 8A), which denoted that PC-NP had a strong glioma targeting effect. Furthermore, the qualitative and quantitative tumor distribution showed that PC-NP achieved the most maximal accumulation in tumor foci (Figure 8A−C). It was speculated that the accumulation of PC-NP was contributed by the neovascular and glioma cells dual targeted ability of Pep-1 and CGKRK peptide. The angiogenetic vasculature recognition and triggered penetration of PC-NP-PTX in glioma site could improve the in vivo antiglioma effect. The group of mice administered PCNP-PTX lived significantly longer than the other groups. Such enhanced antiglioma efficacy of PC-NP-PTX could be probably owing to the Pep-1 and CGKRK modification, which could take advantage of the dual-targeted capacity to improve PTX accumulation at glioma site. The survival time results were consistent with the results of biodistribution. All these data confirmed the hypothesis that PC-NP-PTX could be used as a potential targeted strategy for glioma treatment. Levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CRE) were measured. AST and ALT were used for the evaluation of liver functions,59 and the change in BUN and CRE usually indicated kidney injury.17 According to the data shown in Table S1, there was no significant difference about the AST, ALT, BUN, and CRE levels between Pep-NP, CGKRKNP, PC-NP, and saline group. However, due to the nonspecific uptake by reticuloendothelial system (liver and spleen), both the plain and targeted nanoparticles are unavoidable to expose to MPS. It is reported that only 0.7% (median) of the administered targeting nanoparticle dose is found to be delivered to a solid tumor.60 As we know, Taxol utilizes Cremophor EL as a solubilizer, and Cremophor EL is toxic and can cause life-threatening hypersensitivity reactions. In contrast, in our nanoformulation, PEG-PLGA is a polymer material that can be degraded by organisms and has good biocompatibility, which can reduce the toxicity in comparison to Taxol. It is clear that the dose−response relationship to acute toxicity is needed to clarify the further work. Furthermore, in the future study it is required to investigate the long-term toxic effects of the targeted nanoparticles.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.6b00523. Characterization of nanoparticles; free peptide blocking experiment; antiproliferation assay of blank nanoparticles; in vivo biodistribution of nanoparticles; in vivo safety evaluation and supplementary figures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*(H.X.) Tel: +86-25-86868479. Fax: +86-25-86868467. E-mail: [email protected]. *(Q.X.) Tel: +86-25-86868469. Fax: +86-25-86868469. E-mail: [email protected]. Author Contributions #

These authors contributed equally to this manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported from the National Natural Science Foundation of China (81302710, 81273457, and 31671018), Natural Science Foundation of Jiangsu Province-Excellent Young Scientist Fund (BK20160096), the Ordinary University Natural Science Research Project of Jiangsu Province (13KJB350004), and the Excellent Young Teacher Project of Nanjing Medical University (2015RC16). We are acknowledged the sponsorship of Jiangsu Overseas Research & Training Program for University Prominent Young & Middleaged Teacher and Presidents and 2016 Qing Lan Project of Jiangsu Province.

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ABBREVIATIONS BBB, blood−brain barrier; BTB, blood−tumor barrier; DDS, drug delivery systems; EPR, enhanced permeability and J


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retention; CGKRK, Cys-Gly-Lys-Arg-Lys; IL-13Rα2, interleukin 13 receptor α2; Pep-1, CGEMGWVRC; PTX, paclitaxel; DiR, 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine iodide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FBS, fetal bovine serum; HUVEC, human umbilical vein endothelial cells; TEM, transmission electron microscope; DLS, dynamic light scattering detector; EE, encapsulation efficiency; LC, loading capacity; HPLC, high performance liquid chromatography; PBS, phosphate buffered saline; DAPI, 4′,6-diamidino-2-phenylindole

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