Aptamer-functionalized PEG-PLGA nanoparticles for ...

Biomaterials 32 (2011) 8010e8020

Contents lists available at ScienceDirect

Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Aptamer-functionalized PEGePLGA nanoparticles for enhanced anti-glioma drug delivery Jianwei Guo a, c,1, Xiaoling Gao b,1, Lina Su a, c, Huimin Xia a, Guangzhi Gu a, Zhiqing Pang a, Xinguo Jiang a, Lei Yao b, Jun Chen a, *, Hongzhuan Chen b, * a

Department of Pharmaceutics, Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, PR China Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Road, Shanghai 200025, PR China c College of Pharmacy, Dali University, Wanhua Road, Xiaguan City, Yunnan 671000, PR China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 May 2011 Accepted 4 July 2011 Available online 23 July 2011

Targeted delivery of therapeutic nanoparticles in a disease-specific manner represents a potentially powerful technology especially when treating infiltrative brain tumors such as gliomas. We developed a nanoparticulate drug delivery system decorated with AS1411 (Ap), a DNA aptamer specifically binding to nucleolin which was highly expressed in the plasma membrane of both cancer cells and endothelial cells in angiogenic blood vessels, as the targeting ligand to facilitate anti-glioma delivery of paclitaxel (PTX). Ap was conjugated to the surface of PEGePLGA nanoparticles (NP) via an EDC/NHS technique. With the conjugation confirmed by Urea PAGE and XPS, the resulting Ap-PTX-NP was uniformly round with particle size at 156.0 54.8 nm and zeta potential at 32.93 3.1 mV. Ap-nucleolin interaction significantly enhanced cellular association of nanoparticles in C6 glioma cells, and increased the cytotoxicity of its payload. Prolonged circulation and enhanced PTX accumulation at the tumor site was achieved for Ap-PTX-NP, which eventually obtained significantly higher tumor inhibition on mice bearing C6 glioma xenografts and prolonged animal survival on rats bearing intracranial C6 gliomas when compared with PTX-NP and TaxolÒ. The results of this contribution demonstrated the potential utility of AS1411-functionalized nanoparticles for a therapeutic application in the treatment of gliomas. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Aptamer Nucleolin Nanoparticle Chemotherapy Paclitaxel Drug delivery

1. Introduction Brain tumors remain a significant health problem worldwide, among which glioma is the most commonly diagnosed one, accounting for approximately 45%e50% of all primary brain tumors [1,2]. As an aggressive malignant form of cancer, glioma often results in death of affected patients within one to two years following diagnosis. A defining feature of high-grade glioma (and also seen in lower grade gliomas) is that the tumor does not have a sharp border, individual tumor cells infiltrate the brain, and are likely to be widely distributed at the time of diagnosis [3]. Therefore, malignant brain glioma can rarely be cured with only surgery and radiotherapy. Chemotherapy seems essential in the auxiliary treatment of malignant glioma. However, although has been demonstrated to provide a survival benefit to high-grade glioma * Corresponding authors. Tel.: þ86 21 51980066; fax: þ86 (0) 21 51980069. E-mail addresses: [email protected] (J. Chen), hongzhuan_chen@hotmail. com (H. Chen). 1 Authors contributed equally. 0142-9612/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2011.07.004

patients, the results of chemotherapy have been modest at best [4,5]. Explanations for the poor results include the non-specific, non-targeted nature of most of the drugs currently used and their inadequate delivery to the tumor. Nanobiotechnology, particularly nanoparticles, is making a significant contribution to the improvement of drug delivery in cancer and many of these technologies can be applied to glioma. The challenge lies in the design of nanoparticles (NPs) that are able to overcome the bloodebrain/bloodetumor barrier, specifically and differentially taken up by glioma cells and release their payload over an extended period to achieve a clinical response. NPs derived from poly (D,L-lactic-co-glycolic acid) (PLGA) as the controlled release polymer system are an excellent choice since their safety in clinic is well established [6]. Poly(ethylene glycol) (PEG)-functionalized PLGA NPs are especially desirable because pegylated polymeric NPs have significantly reduced systemic clearance compared with similar particles without PEG [7,8], which is especially important for the passive targeting of nanocarrier to tumor by the enhanced permeability and retention (EPR) effects. The development of biotechnology has provided targeting ligands that

J. Guo et al. / Biomaterials 32 (2011) 8010e8020

specifically bind to biologically active molecules or receptors highly expressed on bloodebrain/bloodeglioma barrier or glioma cells, which further enabled active delivery of chemotherapeutic agents to gliomas [9,10]. Aptamers are single-stranded DNA or RNA oligonucleotides that fold into specific 3D structures, and bind to target molecules with high specificity and affinity. Because of their low molecular weights, lack of immunogenicity, and readily availability, aptamers are good candidates for targeted imaging and therapy. Various aptamers have been developed against a variety of cancer targets, including extracellular ligands and cell surface proteins [11]. AS1411 is a DNA aptamer that binds to nucleolin, a protein highly expressed in the plasma membrane of cancer cells [12], and has been successfully exploited as a targeting ligand for tracking C6 glioma cells [13,14]. It has also been pointed out that nucleolin is highly expressed at the cell surface of endothelial cells in the angiogenic blood vessels [15,16], and participates in binding and endocytosis/macropinocytosis, processes with potential applications in drug delivery [17,18]. Therefore, we speculated that the AS1411-nucleolin interaction could be utilized as a strategy to mediate highly specific and effective drug delivery to gliomas. Paclitaxel (PTX), a widely used chemotherapeutic agent isolated from the bark of Taxus brevifolia, showed anti-neoplasic activity against various types of solid tumors such as ovarian, breast and lung cancers [19,20], and has also been proven effective in the treatment of gliomas [21e24]. However, its clinical efficacy is often compromised by its poor aqueous solubility, non-tumor-specific cell-killing and serious adverse effects induced by its solventdCremophor EL-ethanol [25]. In this study, in order to improve the anti-glioma efficacy of paclitaxel, an AS1411 aptamer-functionalized nanoparticulate drug delivery system (Ap-PTX-NP) was developed. PTX-loaded nanoparticles (PTX-NP) were prepared via an emulsion/solvent evaporation method using PLGAePEGeCOOH amphiphilic copolymer as the matrix. AS1411 was conjugated to PTX-NP surface through

8011

an EDC/NHS technique (Fig. 1). The Ap modification was confirmed by urea polyacrylamide gel electrophoresis (urea PAGE) and X-ray photoelectron spectroscopy (XPS). The resulting nanoparticles were characterized in terms of particle size, zeta potential, surface morphology, drug encapsulation efficiency (EE), drug loading capacity (LC) and in vitro drug release. Cellular association of ApNP and anti-proliferation effect of Ap-PTX-NP was evaluated on C6 glioma cells. Pharmacokinetic and biodistribution were performed to determine the tumor targeting properties of Ap-PTX-NP. In vivo tumor growth inhibition and survival experiment was performed on mice bearing C6 glioma xenografts and rats bearing intracranial C6 gliomas, respectively, to evaluate its anti-glioma efficacy. 2. Materials and methods 2.1. Materials PTX was purchased from Xi’an Sanjiang Biological Engineering Co. Ltd, and TaxolÒ from BristoleMyers Squibb Company. Poly (D,L-lactide-co-glycolide) (50/50) with terminal carboxylate groups (PLGA, inherent viscosity 0.18 dl/g in hexafluoroisopropanol, MW 15 kDa) was obtained from PURCA (Holland). NH2ePEGeCOOH (MW 3500) was purchased from JenKem technology Co. LTD (Beijing, China). AS1411 DNA aptamer (Ap, sequence: 50 -TTGGTGGTGGTG GTTGTGGTGGTGGTGG-30 , 28 bp) was custom synthesized by Sangon Biotech Co., Ltd (Shanghai, China). Both 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and coumarin 6 were provided by SigmaeAldrich (St. Louis, MO, USA). Dubelcco’s Modified Eagle’s Medium (DMEM), trypsineEDTA and penicillinestreptomycin solution were provided by Invitrogen (Merelbeke, Belgium), and fetal bovine serum (FBS) by Gibco BRL (Carlsbad, CA, USA). Double distilled water was purified using a Millipore Simplicity System (Millipore, Bedford, USA). All other reagents were of analytical or chromatographic pure grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Rat C6 glioma cell line was purchased from Cell Institute of Chinese Academy of Sciences (Shanghai, China). SpragueeDawley (SD) rats, Wistar rats and nude mice were obtained from the Shanghai Laboratory Animal Resources Center (Shanghai, China) and treated according to the protocols evaluated and approved by the ethical committee of Fudan University.

Fig. 1. Preparation of aptamer-functionalized paclitaxel-loaded nanoparticles (Ap-PTX-NP). PTX was encapsulated in the PLGAePEGeCOOH nanoparticle via an emulsion/solvent evaporation method. The nanoparticles were decorated with Ap by covalently conjugating amine-terminated Ap to carboxylate-functionalized PTX-NP surface through an EDC/NHS technique. EDC, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide; NHS, N-hydroxysuccinimide.

8012


2.2. Synthesis of PLGAePEGeCOOH copolymer Carboxylate-functionalized copolymer PLGAePEG was synthesized by conjugating COOHePEGeNH2 to PLGAeCOOH according to a previously-described method [26]. The copolymer was dissolved in CDCl3 and characterized by 1H NMR at 400 Hz (Mercury Plus 400, Varian, USA) for determining its number-average molecular weight. 2.3. Preparation of AS1411 aptamer-functionalized paclitaxel-loaded nanoparticles 2.3.1. Preparation of paclitaxel-loaded nanoparticles PTX-NP was prepared with an emulsion/solvent evaporation method as described previously [27]. Firstly, 20 mg PLGAePEG copolymer and 0.4 mg PTX were dissolved in 2 mL dichloromethane, which was then added into a 4 mL of 1% sodium cholate aqueous solution. The mixture was sonicated using a probe sonicator (Scientz Biotechnology Co. Ltd., China) for 30 s at 180 W output. The emulsion formed was added drop-wisely into 20 mL of 0.5% sodium cholate under magnetic stirring. Five minutes later, dichloromethane was evaporated at low pressure and at 30 C using Büchi rotavapor R-200 (Büchi, Germany). The obtained nanoparticles were subjected to a 1.5 10 cm sepharose CL-4B column and eluted with distilled water to remove the unentrapped PTX. Blank nanoparticle (B-NP) was prepared as described above without the addition of PTX. Coumarin 6-labeled nanoparticles were prepared with the same procedure by adding 0.05% (w/w) coumarin 6 for encapsulation, the resulting nanoparticles were subjected to a 1.5 10 cm sepharose CL-4B column and eluted with distilled water to remove the unentrapped coumarin 6. 2.3.2. Conjugation of AS1411 to paclitaxel-loaded nanoparticles surface AS1411 was conjugated to the surface of PTX-NP using an EDC/NHS technique as described by Cheng [26]. PTX-NP was suspended in deionized water (5 mg/mL) and incubated with excess EDC (200 mM) and NHS (100 mM) at RT for 30 min. The resulted N-hydroxysuccinimide-activated PTX-NP was washed with DNase-, RNasefree water (4 mL 4 times) by ultrafiltration (20,000 MWCO, Amicon, Millipore Corporation, Bedford, USA) to remove the residual EDC and NHS. The activated NP was allowed to react with 1 mL of denaturederenatured (85 C for 10 min and snapcooling in ice-water bath for 10 min) 50 -NH2 Ap (0.5 mg/mL) for 6 h under magnetic stirring. The covalently linked Ap-PTX-NP bioconjugates were washed with deionized water (4 mL 4 times) by ultrafiltration, resuspended in distilled water and stored at 4 C until use. 2.4. Characterization of the nanoparticles 2.4.1. Urea polyacrylamide gel electrophoresis For confirming Ap conjugation, NPs treated as described above with (þEDC) or without (EDC) the crosslinker were subjected to 10% Tris/Borate/EDTA (TBE) e Urea PAGE as described previously [26]. Sample sequences loaded on the gel were as follows: PTX-NP, DNA marker, Ap, Ap-PTX-NP (þEDC), Ap-PTX-NP (EDC), washed Ap-PTX-NP (þEDC) and Ap-PTX-NP (EDC). The electrophoresis was performed at 50 V for 160 min, and the base pair (bp) band on the gel was displayed by 0.5 mg/mL ethidium bromide. 2.4.2. X-ray photoelectron spectroscopy X-ray photoelectron spectroscopy (XPS) was performed to measure the surface elemental composition of PTX-NP and Ap-PTX-NP. It was carried out on an RBD upgraded PHI-5000C ESCA system (Perkin Elmer) with Mg Ka(hn ¼ 1253.6 eV)/Al Ka radiation (hn ¼ 1486.6 eV). The X-ray anode was run at 250 W, the high voltage was kept at 14.0 kV, and the detection angle was set at 54 . The pass energy was fixed at 23.5, 46.95 or 93.90 eV to ensure sufficient resolution and sensitivity. The vacuum of the analyzer chamber was lower than 1 108 Torr. The Lyophilized sample was directly pressed to a self-supported disk (10 10 mm), mounted on a sample holder, and then transferred to the analyzer chamber. Both the whole spectra (0e1100 eV) and the narrow spectra for each element with much higher resolution were recorded. With the binding energies calibrated with containment carbon (C1s ¼ 284.6 eV), data analysis was performed using AugerScan 3.21 software. 2.4.3. Particle size and zeta potential Particle size and zeta potential of PTX-NP and Ap-PTX-NP was determined by dynamic light scattering (DLS) analysis using Zeta Potential/Particle Sizer NICOMPÔ 380 ZLS (Santa Barbara, California, USA.) with HeeNe lamp at 632.8 nm. 2.4.4. Surface morphology The morphological examination of PTX-NP and Ap-PTX-NP was performed under transmission electron microscopy (TEM) (H-600, Hitachi, Japan) following negative staining with sodium phosphotungstate solution. 2.4.5. Determination of drug encapsulation efficiency and loading capacity For determining the EE and LC of PTX-NP, a predetermined amount of NPs were dissolved in acetonitrile to release PTX. The PTX level was determined with Agilent 1100 HPLC system (Agilent Technologies, CA, USA) by using a reverse phase C-18 Ultrasphere ODS column (250 4.6 mm, 5 mm, Beckman USA) with methanol and

water (80/20 v/v) as the mobile phase at the flow rate of 1.2 mL/min. The column temperature was maintained at 40 C, sample injection volume was 20 mL and the detection wavelength was 227 nm. 2.4.6. In vitro drug release In vitro PTX release from both PTX-NP and Ap-PTX-NP were performed as described previously [28] using phosphate buffer solution (PBS, pH 7.4) and human plasma as the release media. The nanoparticles were suspended in a centrifuge tube at the concentration of 0.5 mg/mL, which was put in an orbital shaker and vibrated horizontally at 100 rpm and at 37 C. At designated time points (4.5, 15, 24, 48, 72, 96, 144, 168, 192, 216, 240, 264 and 288 h), the tubes were taken out and centrifuged at 11,500 rpm for 45 min with the supernatant subjected to HPLC analysis, and the pellet resuspended in fresh dissolution medium and put back to the shaker for next designated time point measurement. 2.5. Cell experiments 2.5.1. Cell culture C6 cells were cultured in DMEM supplemented with 10% FBS at 37 C in a 5% CO2/95% air humidified environment incubator (Thermo HERAcellÒ, USA). The medium was replenished every other day and the cells were subcultured after reaching confluence. 2.5.2. Cellular association of the nanoparticles Cellular association of Ap-NP was determined quantitatively with High Content Cell Analysis System (HCS) and compared with that of the unmodified nanoparticles using Coumarin 6 as the fluorescent probe as described previously [29]. C6 cells (5000 cells per well) were seeded in a 96-well cell culture plate. Twenty-four hours later, 200 mg/mL Ap-NP (containing coumarin 6 0.09 mg/mL) was added and incubated with the cells at 37 C for 2 h. After that, the cells were washed with D-hanks buffer and fixed with 3.7% formaldehyde solution for 10 min. Stained with 2 mg/mL Hochest 33258 at RT for 1 h away from light, the cell culture plate was washed with PBS for three times and detected under a KineticScanÒ HCS Reader (version 3.1, Cellomics Inc., Pittsburgh, PA, USA). Fluorescent images were captured in the two channels relevant to Hoescht and FITC and analyzed automatically to quantify cell number and cellular associated Ap-NP, respectively. Analysis algorithms were obtained from Cellomics Inc. (Pittsburgh, PA, USA) and referred to as Target Activation. Three thousand cells were detected for each well (n ¼ 3). Specific nature of cellular association of Ap-NP was determined in the presence of excess AS1411 by using the same procedure. 2.5.3. Anti-proliferation assay C6 cells were seeded in 96-well plate at the density of 5000 cells per well and cultured for 24 h to allow cell attachment. After that, the cells were incubated with Taxol, PTX-NP and Ap-PTX-NP respectively at the PTX concentrations of 0.019, 0.038, 0.38, 0.76, 3.8, 12 and 24 mg/mL for 24, 48 and 96 h. Cells treated with Blank Ap-NP were used to determine the anti-proliferative effect of the vehicle and those treated the culture medium were used as negative control. At designated time points, the medium was replaced with DMEM containing MTT (5 mg/mL), and the cells were further incubated at 37 C for 4 h. After that, the MTT solution was removed, 100 mL DMSO was added into each well to dissolve the formazan crystals precipitate. The plate was vigorously shaken for 15 min before the determination of its optical absorbance at 492 nm using a microplate reader (Thermo, USA). The viability of the control cells was taken 100% and used to calibrate that of the treated cells. The experiments were performed in sixplicate. Graphpad Prism statistical analysis software was used for IC50 calculating and plot drawing. 2.6. Pharmacokinetic studies SD rats (200 10 g) were used for the in vivo pharmacokinetic studies. The animals were randomly divided into three groups (n ¼ 4) and intravenously administrated via the tail vein with 3 mg/kg Ap-PTX-NP, PTX-NP and TaxolÒ, respectively. Blood samples were drawn from the carotid vein at 0.1, 0.25, 0.5, 0.75, 1, 2, 4, 8, 12 and 24 h after intravenous administration. The plasma samples were collected following centrifugation at 4000 rpm for 10 min and stored at 20 C until assays. To prepare samples for analysis, 150 mL methanol containing 60 ng/mL docetaxel (internal standard) was added into 50 mL plasma to precipitate the proteins. The mixture was vortexed and subsequently centrifuged at 12000 rpm for 10 min with the supernatant mixed with an equal volume of deionized water and subjected to liquid chromatography-tandem mass spectrometry (LCeMS/MS) analysis. Chromatography was performed using an Agilent 1100 HPLC system with a Gemini C18 column (100 mm 2.0 mm i.d., 3.0 mm, Phenomenex, Torrance, CA, USA) at a temperature of 40 C and a flow rate of 0.3 mL/min using 0.1% formic acid: methanol (3:7) as the mobile phase. Five microliter of sample was injected for analysis. Mass spectrometric detection was performed on an API 3000 triple quadrupole instrument (Applied Biosystems, Toronto, Canada) in Multiple Reaction Monitoring (MRM) mode. A TurboIonSpray ionization (ESI) interface in positive ionization mode was used. Data processing was performed with Analyst 1.4.1 software package (Applied Biosystems). The spray voltage was at 5000 V, ion source

J. Guo et al. / Biomaterials 32 (2011) 8010e8020 temperature at 500 C and collision energy at 30 eV. Detection of the ions was conducted in the multiple reaction monitoring (MRM) mode, monitoring the transition of the m/z 876.6 / 308.0 for paclitaxel (M þ Na)þ and 830.3 / 549.1 for docetaxel (M þ Na)þ, respectively. All the concentration data were dose-normalized and plotted as plasma drug concentrationetime curves. Drug and Statistics software for Windows (DAS ver 2.1.1) was utilized to analyze the pharmacokinetic parameters. 2.7. Tissue biodistribution The tissue biodistribution and tumor targeting properties of Ap-PTX-NP was performed on nude mice bearing glioma xenograft [30,31], and compared with that of PTX-NP and TaxolÒ. The animal model was established by injecting C6 cells (3 107 cells suspended in 150 mL of cell culture medium) subcutaneously in the armpit of right anterior limb. Ten days after the implantation, with the weight of tumors reached about 200 mg, the mice were randomly divided into three groups (28 mice per group) and intravenously administrated with Ap-PTX-NP, PTX-NP and TaxolÒ at a dose of 3 mg/kg PTX, respectively. At predestinate time points (0.25, 0.5, 1, 2, 6, 12 and 24 h) following administration, four mice of each group were sacrificed with the heart, liver, spleen, lung, kidney, brain, and tumor collected and stored at 20 C until analysis. For the determination of PTX, 100 mg of the thawed tissue sample was homogenated in 0.5 mL water with the homogenate pretreated by protein precipitation using docetaxel as the internal standard and subjected to LCeMS/MS analysis as described above.

8013

3.2. Characterization of the fuctionalized nanoparticles 3.2.1. Urea PAGE Urea PAGE was applied to confirm the conjugation of Ap to NP surface (Fig. 3A). NP itself did not show any band on PAGE gel (Fig. 3A, Line. 1). Ap, whose size was 28 bp, showed a band corresponding to a molecular weight smaller than the shortest visible DNA marker (50 bp) (Fig. 3A, Line. 3). In the presence of EDC, Ap was successfully conjugated to NP surface, which was confirmed by the band observed at the loading site (Fig. 3A, Line. 4) as Ap-NP did not migrate under the applied electrophoresis condition. Before washing, a light band corresponding to Ap was visible, indicating the existence of unconjugated Ap. After washing by ultrafiltration, the residual Ap was completely removed (Fig. 3A, Line. 6). In contrast, without the addition of the coupling agent (eEDC), Ap can not be covalently conjugated to NP without showing any band corresponding to Ap-NP at the loading site, but exhibited a thick band corresponding to Ap (Fig. 3A, Line. 5). These data strongly indicated the successful conjugation of Ap to NP surface in the presence of EDC and the complete removal of the unconjugated Ap after washing by ultrafiltration.

2.8. Anti-glioma efficacy In vivo tumor growth inhibition and survival experiments were performed to evaluate the anti-glioma efficacy of the developed formulations. Nude mice model bearing glioma xenograft was established as described in Section 2.7 and used for tumor growth inhibition experiment. Six days after the implantation, with the tumors volume reached about 50 mm3, the mice were randomly divided into four groups (8 mice per group) and intravenously administered with 100 mL Ap-PTX-NP, PTX-NP, TaxolÒ (PTX dose of 3 mg/kg) and saline, respectively. The treatment was repeated every other day for seven consecutive injections. The tumor size was monitored with a caliper every other day, and tumor volume was calculated with the formula: p/6 larger diameter (smaller diameter)2 until the 20th day on which the animals were sacrificed [32]. In order to estimate the adverse effects of the PTX formulations, body weight of the mice was recorded during the treatment. The anti-tumor efficacy of the formulations was also evaluated on Wistar rats bearing intracranial glioma [33,34] by measuring their survival time after treatments. The animal models were set up by implanted C6 cells (3 107 suspended in 10 mL PBS) into the right caudate nucleus (3.0 mm lateral, 2.0 mm anterior to the bregma and 5 mm of depth) by using a stereotaxic instrument (RWD, China). After that, the rats were randomly divided into four groups (6 mice per group) and injected with 100 mL Ap-PTX-NP, PTX-NP, Taxol (PTX dose of 3 mg/kg) and saline via tail vein at 4, 6, 8, 10, 12 and 14 days after implantation. The survival data were presented as KaplaneMeier plots and analyzed with a log-rank test. 2.9. Statistical analysis Unpaired student’s t test was used for between two-group comparison and oneway ANOVA with Fisher’s LSD for multiple-group analysis. A probability (P) less than 0.05 was considered statistically significant. Results were expressed as mean standard deviation (SD) unless otherwise indicated.

3. Results and discussion 3.1. Characterization of PLGAePEGeCOOH copolymer PLGAePEGeCOOH copolymer was synthesized via an EDC/NHS technique. PLGAeCOOH was firstly transformed into PLGAeNHS, to obtain and then reacted with NH2ePEGeCOOH PLGAePEGeCOOH. The chemical composition of the synthesized product was confirmed by 1H NMR (Fig. 2). The characteristic peaks at 1.55, 4.8 and 5.2 ppm belonged to the methyl (d,eCH3), methene (m,eCH2) and methine (m,eCH) proton of PLGA segment respectively, and the peak at 3.6 ppm belonged to the methene (s,eCH2) proton of PEG chain. By using the relative molecular weights and the integration of characteristic peaks at 5.20 and 3.6 ppm [26], number-average molecular weight of the synthesized copolymer was calculated to be 22,800, the conjugation efficiency of NH2ePEGeCOOH to PLGAeCOOH was estimated to be 25%, and the yield of the reaction was determined to be 35%.

3.2.2. X-ray photoelectron spectroscopy The surface chemical compositions of PTX-NP and Ap-PTX-NP were identified by XPS analysis (Table 1). The C1s spectra were composed of four peaks at 285.0, 286.8, 287.6 and 289.4 eV, respectively, where the peak at 286.8 eV mainly represented eCeOeC groups of the PEG component on the nanoparticle surface. The decomposition of the O1s envelope revealed the presence of two types of oxygen: O]C at 532.7 eV and OeC at 533.6 eV. The peak at 132 eV was attributed to P2p3 envelope that was only detected in Ap-PTX-NP with a value of 0.1% with regard to the total amount of C, O, N and P atoms. According to the chemical composition of the samples, the phosphorus could only be ascribed to Aptamer, which confirmed the decoration of aptamer on the nanoparticle surface. 3.2.3. Particle size and polydispersity Under the optimized condition (sonication power 180 W, sonication time 30 s, the ratio of PTX to PLGAePEG copolymers 2%, and the concentration of sodium cholate 1.5%), the mean diameter of PTX-NP was 121.0 35.8 nm (P.I. ¼ 0.088). A slight increase in diameter was observed following its modification with Ap (156.0 54.8 nm, P.I. ¼ 0.123). 3.2.4. Zeta potential The zeta potential of PTX-NP and blank NP was observed to be 23.70 2.4 mV and 24.14 1.9 mV (n ¼ 3), respectively, which indicated that the encapsulation of PTX did not influence the surface charge of the nanocarrier. Following the conjugation with Ap, the zeta potential of PTX-NP was reduced to 32.93 3.1 mV (n ¼ 3). 3.2.5. Surface morphology Surface morphology of PTX-NP and Ap-PTX-NP was investigated by TEM (Fig. 3B). Both nanoparticles were in moderate uniformity and showed a spherical shape and smooth surface. The sphere volume of Ap-PTX-NP was slightly larger than that of PTX-NP. The particle size observed by TEM was similar but slightly smaller than the values obtained from the DLS method. 3.2.6. Drug encapsulation efficiency and loading capacity The EE of the optimized PTX-NP and Ap-PTX-NP formulation was 49.6 4.7% and 44.7 3.9% (n ¼ 3), respectively. And the LC of PTX-NP and Ap-PTX-NP was 1.32 0.05% and 1.02 0.08% (n ¼ 3),

8014


respectively. Sonication power and the feeding ratio of PTX to PLGAePEG copolymers were found to be the two key factors that affected EE and particle size (Table 2). By increasing the sonication power from 80 to 200 W, EE was decreased from 61.7 3.9 to 43.2 4.6% (n ¼ 3). By increasing the PTX feeding ratio from 1.0% to 5.0%, EE was decreased from 65.7 4.9 to 43.6 2.4% (n ¼ 3).

reached 62.9 2.5% and 60.6 4.9% in PBS, 67.2 2.9% and 64.2 4.6% in plasma for PTX-NP and Ap-PTX-NP, respectively. After that, both PTX-NP and Ap-PTX-NP exhibited sustained release with the total release in 12 days reaching 79.4 3.8% and 78.4 4.5% in PBS, 87.0 4.2% and 84.1 5.8% in plasma for PTX-NP and Ap-PTX-NP, respectively.

3.2.7. In vitro drug release In vitro PTX release from PTX-NP and Ap-PTX-NP were investigated in pH 7.4 PBS and human plasma at 37 C. The two formulations exhibited similar release patterns in both release media (Fig. 3C). A fast release of PTX from PTX-NP/Ap-PTX-NP was observed in the initial 24 h (PTX-NP: 44.7 4.4% in PBS and 47.8 6.4% in plasma; Ap-PTX-NP: 43.3 3.3% in PBS and 46.8 2.7% in plasma). In the following 48 h, the cumulative release

3.3. Cell experiments 3.3.1. Cellular association Cellular association of Ap-NP was determined by HCS analysis using coumarin 6 as the fluorescent probe. As showed in Fig. 4, cellular association of Ap-NP was significantly higher than that of the unmodified NPs (about 2 folds), and greatly reduced in the presence of excess AS1411.

Fig. 2. 1H NMR spectrum of (A) H2NePEGeCOOH, (B) PLGAeCOOH and (C) the synthesized PEGePLGAeCOOH copolymer in CDCl3. Characteristic peaks as marked in the graphs.


8015

3.3.2. Anti-proliferation assay The effect of TaxolÒ, PTX-NP and Ap-PTX-NP on cell viability was evaluated by MTT assay. The range of PTX concentrations (0.019e24 mg/mL) was set corresponding to the plasma PTX levels achievable in human [35]. Apparent growth suppression on C6 glioma cells was observed after PTX treatment in a concentration, time and formulation-dependent pattern. As showed by IC50, which was defined as the drug concentration at which 50% cells were inhibited in growth or survival (Table 3), anti-proliferation ability of the formulations followed the order: Ap-PTX-NP > PTXNP > TaxolÒ. PTX-NP showed slightly higher cytotoxicity over TaxolÒ especially after long-time incubation (48 and 96 h). Significantly enhanced cytotoxicity (IC50 2.59, 4.18 and 4.67 times lower than that of TaxolÒ and 2.92, 3.32 and 2.67 times lower than that of PTX-NP after 24, 48 and 96 h treatment, respectively) was achieved for Ap-PTX-NP. 3.4. Pharmacokinetic study After intravenous administration of TaxolÒ, PTX-NP and Ap-PTXNP at the dose of 3 mg/kg PTX, plasma PTX concentrationetime profiles were shown in Fig. 5. In consistent with previous studies [36,45,46], PTX was rapidly cleared from the systemic circulation following intravenous administration of its solution formulationdTaxolÒ. In contrast, following its encapsulation in NPs (both Ap-PTX-NP and PTX-NP), much higher concentration and longer circulation time was achieved. The pharmacokinetic profile of PTX following intravenous administration was found to fit a twocompartment model and the pharmacokinetic parameters were calculated with DAS Version 2.1.1. As showed in Table 4, both PTXNP and Ap-PTX-NP showed significant longer elimination half life (t1/2b), slower clearance rate (CL), smaller elimination rate constant (K10) and higher AUC (5.8 and 5.4 folds, respectively, P < 0.01) when compared with TaxolÒ. As we compared the differences in pharmacokinetic profiles between PTX-NP and Ap-PTX-NP, although not significant different, we found that the clearance of Ap-PTX-NP was slightly faster than that of PTX-NP, and the AUC of Ap-PTX-NP was slightly smaller than that of PTX-NP (Table 4). 3.5. Tissue biodistribution Tissue biodistribution of PTX following intravenous administration of TaxolÒ, PTX-NP and Ap-PTX-NP were assessed in nude mice bearing glioma xenograft. PTX was found to be distributed widely and rapidly into various organs (heart, liver, spleen, kidney, lung and tumor) after intravenous administration of the three formulations (Fig. 6, data not shown for other time points). The highest PTX concentration was found in the liver and followed the order: Ap-PTX-NP > PTX-NP > TaxolÒ. In other organs, PTX distribution followed the order: kidney w spleen > lung > heart > tumor > brain. At all time points, PTX concentrations determined in the tumors of those animals received the three formulations followed the order: Ap-PTX-NP > PTX-NP > TaxolÒ (Fig. 6C). Specifically, 0.25, 0.5, 1, 2, 6, 12 and 24 h after administration, PTX level in the tumor of Ap-PTX-NP group were 1.07, 1.37, 1.72, 1.43, 1.38, 2.41 and 1.57fold (P < 0.05) over that of PTX-NP group, and 1.36 (P > 0.05), 2.32, 1.97, 2.41, 2.83, 3.70 and 2.44-fold (P < 0.05) over that of TaxolÒ group, respectively. 3.6. In vivo anti-glioma efficacy In vivo anti-tumor efficacy of Ap-PTX-NP was evaluated in mice bearing glioma xenograft and compared with that of TaxolÒ and

Fig. 3. Characterization of AS1411 aptamer-functionalized nanoparticles. (A) Confirmation of the conjugation of aptamer (Ap) to PEGePLGA nanoparticles (NP) via urea polyacrylamide gel electrophoresis. Bands corresponding to Ap, Ap-NP and DNA marker as indicated by arrows. In the presence of EDC, Ap was conjugated to NP surface with a band observed at the loading site. After washed by ultrafiltration, the unconjugated Ap was completely removed; (B) TEM images of PTX-NP (A and C) and Ap-PTX-NP (B and D). NPs were negatively stained with phosphotungstic acid solution. Bar, 0.5 mm and 100 nm, respectively, as shown in the figure; (C) In vitro release profile of Paclitaxel from PTX-NP and Ap-PTX-NP in pH 7.4 PBS and plasma, respectively (n ¼ 3).

PTX-NP. The mice were intravenously administered with TaxolÒ, PTX-NP, Ap-PTX-NP (PTX dose of 3 mg/kg) every 2 days for seven consecutive injections with tumors size recorded every other day until the 20th day (Fig. 7). Animals received saline was used as

8016


Table 1 X-ray photoelectron spectroscopy (XPS) analysis of PTX-NP and Ap-PTX-NP. Samples

XPS elemental ratio (%) C

XPS C1s envelope ratios (%)

O

N

P

CeC/CeH

CeOeC

XPS O1s envelope ratios (%) CeOeC]O

OeC]O

O]C

OeC

Binding energy (eV)

PTX-NP Ap-PTX-NP

61.44 61.62

38.24 37.90

0.31 0.38

e 0.10

285.00

286.80

287.60

289.40

532.73

533.62

32.9 32.8

28.1 28.2

21.4 21.4

17.6 17.6

56.9 57.6

43.1 42.4

eUnder detection limit.

control. In the initial six days, no obvious differences in the tumor size were observed among the groups. Following that, from the 8th to 20th day, the tumor volume of the animals received the PTX formulations followed the order: Ap-PTX-NP < PTXNP < Taxol < Saline. At the experimental terminal, the tumor sizes of the PTX-treated groups were all notably smaller than that of the saline group (Fig. 8), and followed the same order: Ap-PTXNP < PTX-NP < Taxol < Saline. Based on tumor volume and weight, the average tumor inhibition of Ap-PTX-NP, PTX-NP and TaxolÒ were calculated to be 81.68% and 79.93%, 66.95% and 68.69%, and 46.75% and 48.25%, respectively. In order to estimate the adverse effects of the PTX formulations, body weight of the mice was recorded during the treatment. From the 1st to 10th day, the average body weight of the mice in all groups showed slow increase without significant difference among the groups. Following that, the average body weight of the saline group decreased gradually, and reduced 14.33% of their initial weight by the experimental terminal. But in the case of the TaxolÒ, PTX-NP and Ap-PTX-NP groups, the body weight was increased by 9.51%, 11.56% and 17.89%, respectively, significant higher than that of the saline group (P < 0.01). The anti-glioma efficacy of the formulations was also evaluated by the survival experiment performed on Wistar rats bearing intracranial C6 gliomas. The median survival time of the animals treated with saline, TaxolÒ, PTX-NP and Ap-PTX-NP were 18, 24, 27 and 31 days, respectively (Fig. 9). Ap-PTX-NP significantly prolonged animal survival when compare with PTX-NP (22%) (P < 0.05), TaxolÒ (39%) (P < 0.01) and saline (72%) (P < 0.001). The survival time of the PTX-NP group was also significantly longer than that of the TaxolÒ one (P < 0.05).

4. Discussion Treatment of glioma, a primary malignant tumor of the brain, is one of the most challenging problems in healthcare as no currently available treatment is curative. Surgery remains the basic treatment in which the bulk of the tumor is removed and the peripheral infiltrating part is the target of supplementary treatments. The currently available anti-cancer therapeutics are less than optimal for glioma treatment, mainly owing to the delivery problems.

Table 2 Effects of sonicating power and feeding ratio of PTX to PLGAePEG on the encapsulation efficiency (EE) and diameter of PTX-NP (n ¼ 3). Effect factors

Level

EE (%)

Diameter (nm)

Sonication power (W)

80 140 200

61.7 3.9 52.7 2.3 43.2 4.6

193.5 8.2 154.6 6.8 98.3 8.4

65.7 4.9 53.4 3.3 43.6 2.4

96.5 7.6 112.2 5.5 138.3 4.8

Feeding ratio of PTX to PLGAePEG (%)

1.0 3.0 5.0

Nanobiotechnology approaches where a constant dose of chemotherapy is delivered directly to cancer cells provide alternative or complementary therapeutic options for patients suffering from gliomas. Nanoparticulate drug delivery strategies such as bloodebrain barrier overcoming [37], glioma cells targeting [38] and bloodebrain barrier and glioma dual-targeting [9,10] have been exploited for elevated drug delivery to glioma. In this contribution, AS1411, a DNA aptamer specifically binding to nucleolin, was utilized as the targeting ligand to functionalize a biocompatible nanoparticulate DDS for anti-glioma drug delivery. Nucleolin is a protein highly expressed in the plasma membrane of both cancer cells and endothelial cells in angiogenic blood vessels. As angiogenesis is one of the most important events for tumor growth, evasion and metastasis, it was speculated that the AS1411nucleolin interaction-mediated glioma-targeting drug delivery would be more specific and effective than those bloodebrain barrier overcoming strategies. The aptamer was conjugated to the surface of PEGePLGA nanoparticles using an EDC/NHS technique as described by Cheng [26] and Farokhzad [39]. The conjugation was confirmed by Urea PAGE, XPS and zeta potential measurement. The observed band corresponding to Ap-PTX-NP at the loading site under Urea PAGE and the detected phosphorus binding energy under XPS strongly suggested an effective Ap decoration. In the case of zeta potential, consistent with a previous study [39], the presence of carboxymodified PEG on the NP surface resulted in a negative surface charge, which might provide advantages such as decreasing the non-specific interaction between the negative charged Ap and NP surface, thus preserving Ap conformation and binding characteristics. The reduction in zeta potential of PTX-NP following its modification with the negative charged Ap further confirmed the conjugation. Particle size is one of the most important parameters that determines the fate of both in vitro and in vivo nanoparticulate DDS [7,40]. It has been pointed out that nanocarriers between 100 and 200 nm are known to have favorable EPR effect within tumor vasculature [41]. In this contribution, an orthogonal experimental design was utilized to optimize the preparation procedure using particle size and PTX encapsulation efficiency as the response variables. Under the optimized condition, the mean diameter of PTX-NP and Ap-PTX-NP were in the range for optimal EPR effects. It has been pointed out that the increase of drug payload might increase NP polydispersity [26]. In this study, both PTX-NP and ApPTX-NP exhibited a narrow particle distribution (PI < 0.13) [42], which indicated that a nice encapsulation of PTX was achieved. PTX release from both PTX-NP and Ap-PTX-NP underwent a biphases release pattern. The burst release of PTX in the first 12 h was believed to be contributed by the agent that was poorly entrapped and located at the periphery of PLGA matrix, while the sustained release in the following days was mainly attributed to the diffusion of drug that was well encapsulated and localized in the NP core. Although no significant differences were detected in both release media at all time points, the cumulative PTX release from


8017

Fig. 4. Cellular association of AS1411-functionalized nanoparticles (Ap-NP) in C6 glioma cells as showed by a high content cell analysis system using coumarin-6 as the fluorescence probe. (A) Unmodified nanoparticles (NP); (B) Ap-NP; (C) Ap-NP in the presence of excess Ap; (D) Quantitation of cellular association of the nanoparticles in C6 glioma cells. Values obtained from 3000 cells per well (n ¼ 3) and expressed as mean SD. **P < 0.01, significantly lower than the cellular association of coumarin-6-loaded Ap-NP.

PTX-NP was slightly higher than that from Ap-PTX-NP, which we believed was due to the larger size of Ap-PTX-NP following Apconjugation. From both PTX-NP and Ap-PTX-NP, PTX release in plasma was slightly higher than that in PBS, which might be resulted from the enhanced matrix erosion in plasma. As shown by HCS analysis, cellular association of Ap-NP was significantly higher than that of the unmodified NPs, but greatly reduced in the presence of excess AS1411, which strongly suggested that cellular association of Ap-NP was facilitated by the specific interaction between nucleolin and the aptamer AS1411 [13,43]. The anti-proliferation IC50 obtained for TaxolÒ and PTX-NP was consistent with previous studies [21,24,44]. PTX-NP showed slightly higher cytotoxicity over TaxolÒ especially after a long incubation period (48 and 96 h), which was probably due to the enhanced stability and sustained release of PTX following its encapsulation in NP. Significantly enhanced cytotoxicity was achieved for Ap-PTX-NP (IC50 2.59, 4.18 and 4.67 times lower than that of TaxolÒ and 2.92, 3.32 and 2.67 times lower than that of PTX-NP after 24, 48 and 96 h treatment, respectively), which was even more effective than the well-known integrin-mediated or low density lipoprotein receptor-mediated tumor-cell targeting strategies [21,24]. In addition, it was found that blank Ap-NP did not show apparent anti-proliferative activity at our experimental time points (data not shown). Therefore, we believed that the enhanced cytotoxicity of Ap-PTX-NP was mainly owed to its elevated cellular association and uptake facilitated by the nucleolin-AS1411 interaction. As nucleolin is a protein highly expressed on the cell surface where it serves as a binding protein for a variety of ligands implicated in tumorigenesis and angiogenesis, and emerging evidence suggests that the cell-surface expressed nucleolin is a strategic target for an effective and nontoxic cancer therapy [12], we believed that the nucleolin-AS1411 interaction could be utilized as an effective tumor-targeting drug delivery strategy. Consistent with previous pharmacokinetic studies [36,45,46], PTX was rapidly cleared from the systemic circulation following intravenous administration of its solution formulationdTaxolÒ (clearance half life of 1.79 h). In contrast, following its

encapsulation in NPs (both Ap-PTX-NP and PTX-NP), much higher concentration and longer circulation time (half life of 4.16 h and 5.43 h for Ap-PTX-NP and PTX-NP, respectively) was achieved, which was believed to be attributed to its enhanced stability and the stealth effects of pegylated nanoparticles [47]. However, it was noticed that the circulation half life of NPs we obtained was similar with that reported by Xu et al. [46] but seemed relatively short compared to the data obtained by Yu et al. [47]. We speculated that this mainly resulted from the shorter PEG length (3000 Da) and lower NP dose (about 100 mg/kg) that we used since both previous studies and our work (unpublished data) indicated that PEG length, particle size [7] as well as NP doses [48] could influence the in vivo behavior of NPs. Although there was no statistical significance detected, the clearance of Ap-PTX-NP was found slightly faster than that of PTXNP, and the AUC of Ap-PTX-NP was also slightly smaller than that of PTX-NP (Table 4). We speculated that this resulted from its surface Ap decoration, which may partly compromise the stealth effect of PEG. This hypothesis was confirmed by the fact that Ap-PTX-NP showed slightly higher PTX accumulation than PTX-NP in the organs of MPSdthe liver and the spleen (Fig. 6). These data were in good consistency with the results of Gu et al. which showed that NP-Ap 10% exhibited significantly higher particle accumulation in the liver compared to NPs with a lower Ap surface density (0%, 1% and 5%) while NP-Ap 5% showed the highest accumulation in the tumor [49]. This telling evidence together indicated that in engineering targeted NPs, one must balance the tumor-targeting ligand surface density and the surface stealth properties to optimize their in vivo pharmacokinetics. Xenograft tumors are characterized by synchrony and reproducibility of tumor formation, rapid tumor development, and high

Table 3 IC50 value of TaxolÒ, PTX-NP and Ap-PTX-NP on C6 glioma cell line following 24, 48 and 96 h treatment, respectively (n ¼ 6). Time (h)

24 48 96

IC50 (mg/mL) Taxol

PTX-NP

Ap-PTX-NP

29.8 2.3 11.7 1.5 0.14 0.03

33.6 2.8 9.3 2.1 0.08 0.02*

11.5 1.7***/### 2.8 0.5***/## 0.03 0.01**/#

Significant differences between Ap-PTX-NP/PTX-NP and TaxolÒ was marked as *p < 0.05, **p < 0.01, ***p < 0.001; between Ap-PTX-NP and PTX-NP was labeled with #p < 0.05, ##p < 0.01, ###p < 0.001.

Fig. 5. PTX concentrationetime profile following intravenous administration of TaxolÒ (blue), PTX-NP (pink) and Ap-PTX-NP (green) in SD rats at the dose of 3 mg/kg (n ¼ 4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

8018


Table 4 Pharmacokinetic parameters of PTX following intravenous administration of TaxolÒ, PTX-NP and Ap-PTX-NP at the dose of 3.0 mg/kg (n ¼ 4). Parameters

TaxolÒ

PTX-NP

Ap-PTX-NP

t1/2a (h) t1/2b (h) V1 (L) CL (L/h) AUC(0t) (mg/L h) AUC(0N) (mg/L h) K10 (1/h) K12 (1/h) K21 (1/h)

0.05 1.79 0.03 0.09 918.78 1018.34 2.61 8.44 1.54

0.61*** 5.43* 0.06 0.01*** 5351.30*** 5681.46*** 0.28*** 0.51*** 0.45**

0.44*** 4.16* 0.05 0.01*** 4982.11*** 5235.47*** 0.35*** 0.70*** 0.66*

Significant differences was found between the Ap-PTX-NP/PTX-NP and TaxolÒ group and marked as *p < 0.05, **p < 0.01, ***p < 0.001. No significant difference was found between the Ap-PTX-NP and PTX-NP group.

Fig. 6. Tissue biodistribution of PTX after intravenous administration of TaxolÒ (blue), PTX-NP (pink) and Ap-PTX-NP (green) to mice bearing C6 glioma xenograft at the dose of 3.0 mg/kg (A) 0.25 and (B) 2 h following administration; (C) PTX concentrations in the tumor at 0.25, 0.5, 1, 2, 6, 12 and 24 h after administration (n ¼ 4). Significant differences found between the Ap-PTX-NP/PTX-NP and the TaxolÒ group, and marked as *p < 0.05, **p < 0.01, ***p < 0.001 respectively; Significant differences found between the Ap-PTX-NP and the PTX-NP groups, and labeled with #p < 0.05, ##p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

penetrance. The use of s.c. models allows for easy tumor visualization, making decisions of treatment initiation and drug application [50] and have been widely applied in anti-cancer drug development for various cancers including prostate cancer [51], hepatic cancer [52], lung cancer [53] and glioma [30,31]. In this contribution, xenograft nude mouse models with glioma implanted in the armpit were used for determining in vivo drug distribution and tumor growth delay, which provided convenience in tumor separation and visualization, and served as an important complementarity to the intracranial tumor model. Biodistribution study in xenograft nude mouse models showed that at all time points, PTX concentrations determined in the tumors of those animals received the three formulations in the following order: Ap-PTX-NP > PTX-NP > TaxolÒ. The different tumor PTX disposition was contributed by the different formulation design. TaxolÒ, PTX solution for injection, formulated in a mixed solvent composed of Cremophor EL and ethanol 1:1 (v/v), was not designed as tumor targeting formulation and showed the lowest tumor PTX disposition. PTX-NP, due to its favorable EPR effects, showed higher drug accumulation at the tumor site. Ap-PTX-NP, functionalized with Ap which has its specific receptor highly expressed in the plasma membrane of both glioma cells and endothelial cells in angiogenic blood vessels, showed the highest PTX accumulation at the tumor site. For evaluating the anti-glioma efficacy, our dosing schedule on the xenograft nude mouse models was set according to those schedules usually used in anti-glioma therapy (Total dose: 15e250 mg/kg, every 2e3 days a dose) [21,23,54]. A relatively low dose (21 mg/kg in total, 3 mg/kg per dose, every 2 days a dose for seven consecutive injections) was used in this study as our preliminary experiment showed that high PTX dose (especially the TaxolÒ group) could result in high toxicity to the tumor-bearing animals. Under this dosing schedule, the anti-tumor efficacy was easily observed while the toxicity remained minimal as evidenced by the increasing body weight of the animals during the experimental period. As shown in Fig. 7, tumor growth delay of Ap-PTXNP was found after only one dose (Day 8), and became significant after only 3 doses (Day 12) compared to other groups. Intracranial tumor model in Wistar rats was utilized as another animal model for efficacy evaluation by providing survival data. The dosing schedule applied for the intracranial tumor model was adopted from that used in the xenograft mouse model. Although

Fig. 7. Tumor growth curve on nude mice bearing C6 glioma xenograft after intravenous administration of TaxolÒ (blue), PTX-NP (pink), Ap-PTX-NP (green) or saline (yellow) at the dose of 3.0 mg/kg via the tail vein every other day during a 20 days period (n ¼ 8). Significant differences found between the Ap-PTX-NP/PTX-NP and the TaxolÒ groups, and marked as *p < 0.05, **p < 0.01, ***p < 0.001 respectively; Significant differences found between the Ap-PTX-NP and the PTX-NP groups, and labeled with #p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


8019

Fig. 9. KaplaneMeier survival log-rank analysis of Wistar rats bearing intracranial C6 glioma (n ¼ 6). The survived time of the animals received Ap-PTX-NP was significantly longer than that of those received PTX-NP (P < 0.05), TaxolÒ (P < 0.01) and saline (P < 0.001).

Fig. 8. Anti-tumor efficacy of TaxolÒ, PTX-NP and Ap-PTX-NP on nude mice bearing glioma xenograft at the experiment terminal (n ¼ 8). (A) Tumor xenografts alignment of each group taken out from the sacrificed mice at the study end point. (B) Tumor volume at the study end point. (C) Tumor weight at the study end point. Significant differences found between the Ap-PTX-NP/PTX-NP and the TaxolÒ groups, and marked as *p < 0.05, ***p < 0.001 respectively; Significant differences found between the ApPTX-NP and the PTX-NP groups, and labeled with #p < 0.05.

the increased survival time of Ap-PTX-NP compared to nontargeted NPs, TaxolÒ and saline was only 4, 7 and 13 days, it was significant in both biological value and statistical differences (ApPTX-NP vs PTX-NP, [22%, P < 0.05; Ap-PTX-NP vs TaxolÒ, [39%, P < 0.01; Ap-PTX-NP vs saline, [72%, P < 0.001). This anti-glioma effect of Ap-PTX-NP reported here was superior to that of previous studies using Angiopep-2 [23], M-PEG-NP [55] or RGD-NP [21] to mediate PTX treatment in intracranial glioma model. Although it is still controversial about the destructive degree of BBB during the occurrence of glioma [56], strategies overcoming BBB [37], targeting tumor cells [38] and dual-targeting BBB and glioma [9,10] have shown enhanced drug delivery to glioma, suggesting that BBB could partly but not completely hinder antiglioma drug delivery. The potential disadvantage of those strategies overcoming BBB is their non-specific distribution in the central nervous system (CNS), which might cause severe adverse effects therein. The drawback of tumor-targeting procedures was their lack of BBB-overcoming property, which might result in poor drug accumulation in glioma with intact BBB. As angiogenesis is one of the most important characteristics for tumor growth, evasion and metastasis, it was speculated that angiogenesis vascular and tumor

cells dual-targeting strategies might provide more specific and effective anti-glioma drug delivery. In this contribution, the target receptordnucleolin was highly expressed in the plasma membrane of both cancer cells and endothelial cells in angiogenic blood vessels, therefore the drug delivery strategy described here met the requirements of angiogenesis vascular and tumor cells dualtargeting and has shown excellent anti-glioma effects both in vitro and in vivo. Although the relative contribution of angiogenesis vascular targeting and tumor cells targeting to the total anti-glioma drug delivery was not fully disclosed here, the results of this study strongly verified the application of AS1411-modified PTX-NP in inhibiting in situ glioma growth and prolonging animal survival. Further studies will be performed to illuminate the mechanisms of how AS1411 facilitate PTX-NP delivery to glioma in the CNS. 5. Conclusion The present study developed a nanoparticulate DDS decorated with AS1411 aptamer as the targeting ligand for anti-glioma drug delivery. The aptamer were conjugated to the surface of PEGePLGA nanoparticles using an EDC/NHS technique. The conjugation was confirmed by Urea PAGE and XPS. The resulted Ap-PTX-NP was observed to be uniformly spherical in shape with a particle size of 156.0 54.8 nm and zeta potential of 32.93 3.1 mV. The AS1411-nucleolin mediated recognition and internalization significantly facilitated the cellular association of Ap-NP in C6 glioma cells, and improved the cytotoxicity of its payload. The longcirculating property and AS1411 ligand of Ap-PTX-NP warranted rapid, long-term, and accurate in vivo tumor targeting, and improved anti-glioma efficacy for PTX on mice bearing C6 glioma xenograft and rats bearing intracranial C6 gliomas. Acknowledgment This work was supported by National Natural Science Foundation of China (30801439, 81072592, 30801442), National Key Basic Research Program (2007CB935800, 2010CB529800) and Grants from Shanghai Science and Technology Committee Rising-Star Program (10QA1404100).

8020


References [1] Wrensch M, Minn Y, Chew T, Bondy M, Berger MS. Epidemiology of primary brain tumors: current concepts and review of the literature. Neuro Oncol 2002;4:278e99. [2] Behin A, Hoang-Xuan K, Carpentier AF, Delattre JY. Primary brain tumours in adults. Lancet 2003;361:323e31. [3] Claes A, Idema AJ, Wesseling P. Diffuse glioma growth: a guerilla war. Acta Neuropathol 2007;114:443e58. [4] Hart MG, Grant R, Garside R, Rogers G, Somerville M, Stein K. Chemotherapy wafers for high grade glioma. Cochrane Database Syst Rev 2011;3:D7294. [5] Pipas JM, Meyer LP, Rhodes CH, Cromwell LD, McDonnell CE, Kingman LS, et al. A phase II trial of paclitaxel and topotecan with filgrastim in patients with recurrent or refractory glioblastoma multiforme or anaplastic astrocytoma. J Neurooncol 2005;71:301e5. [6] Langer R. Drug delivery and targeting. Nature 1998;392:5e10. [7] Alexis F, Pridgen E, Molnar LK, Farokhzad OC. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharmacol 2008;5:505e15. [8] Gref R, Minamitake Y, Peracchia MT, Trubetskoy V, Torchilin V, Langer R. Biodegradable long-circulating polymeric nanospheres. Science 1994;263: 1600e3. [9] Xin H, Jiang X, Gu J, Sha X, Chen L, Law K, et al. Angiopep-conjugated poly(ethylene glycol)-co-poly(epsilon-caprolactone) nanoparticles as dualtargeting drug delivery system for brain glioma. Biomaterials 2011;32: 4293e305. [10] He H, Li Y, Jia XR, Du J, Ying X, Lu WL, et al. PEGylated poly(amidoamine) dendrimer-based dual-targeting carrier for treating brain tumors. Biomaterials 2011;32:478e87. [11] Barbas AS, Mi J, Clary BM, White RR. Aptamer applications for targeted cancer therapy. Future Oncol 2010;6:1117e26. [12] Hovanessian AG, Soundaramourty C, El KD, Nondier I, Svab J, Krust B. Surface expressed nucleolin is constantly induced in tumor cells to mediate calciumdependent ligand internalization. PLoS One 2010;5:e15787. [13] Hwang DW, Ko HY, Lee JH, Kang H, Ryu SH, Song IC, et al. A nucleolin-targeted multimodal nanoparticle imaging probe for tracking cancer cells using an aptamer. J Nucl Med 2010;51:98e105. [14] Ko HY, Choi KJ, Lee CH, Kim S. A multimodal nanoparticle-based cancer imaging probe simultaneously targeting nucleolin, integrin alphavbeta3 and tenascin-C proteins. Biomaterials 2011;32:1130e8. [15] Fogal V, Sugahara KN, Ruoslahti E, Christian S. Cell surface nucleolin antagonist causes endothelial cell apoptosis and normalization of tumor vasculature. Angiogenesis 2009;12:91e100. [16] Christian S, Pilch J, Akerman ME, Porkka K, Laakkonen P, Ruoslahti E. Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels. J Cell Biol 2003;163:871e8. [17] Reyes-Reyes EM, Teng Y, Bates PJ. A new paradigm for aptamer therapeutic AS1411 action: uptake by macropinocytosis and its stimulation by a nucleolin-dependent mechanism. Cancer Res 2010;70:8617e29. [18] Legrand D, Vigie K, Said EA, Elass E, Masson M, Slomianny MC, et al. Surface nucleolin participates in both the binding and endocytosis of lactoferrin in target cells. Eur J Biochem 2004;271:303e17. [19] Rowinsky EK. Clinical pharmacology of taxol. J Natl Cancer Inst Monogr; 1993:25e37. [20] Hajek R, Vorlicek J, Slavik M. Paclitaxel (Taxol): a review of its antitumor activity in clinical studies minireview. Neoplasma 1996;43:141e54. [21] Zhan C, Gu B, Xie C, Li J, Liu Y, Lu W. Cyclic RGD conjugated poly(ethylene glycol)-co-poly(lactic acid) micelle enhances paclitaxel anti-glioblastoma effect. J Control Release 2010;143:136e42. [22] Son MJ, Song HS, Kim MH, Kim JT, Kang CM, Jeon JW, et al. Synergistic effect and condition of pegylated interferon alpha with paclitaxel on glioblastoma. Int J Oncol 2006;28:1385e92. [23] Regina A, Demeule M, Che C, Lavallee I, Poirier J, Gabathuler R, et al. Antitumour activity of ANG1005, a conjugate between paclitaxel and the new brain delivery vector Angiopep-2. Br J Pharmacol 2008;155:185e97. [24] Nikanjam M, Gibbs AR, Hunt CA, Budinger TF, Forte TM. Synthetic nano-LDL with paclitaxel oleate as a targeted drug delivery vehicle for glioblastoma multiforme. J Control Release 2007;124:163e71. [25] Singla AK, Garg A, Aggarwal D. Paclitaxel and its formulations. Int J Pharm 2002;235:179e92. [26] Cheng J, Teply BA, Sherifi I, Sung J, Luther G, Gu FX, et al. Formulation of functionalized PLGA-PEG nanoparticles for in vivo targeted drug delivery. Biomaterials 2007;28:869e76. [27] Gao X, Tao W, Lu W, Zhang Q, Zhang Y, Jiang X, et al. Lectin-conjugated PEGePLA nanoparticles: preparation and brain delivery after intranasal administration. Biomaterials 2006;27:3482e90. [28] Zhang Z, Feng SS. The drug encapsulation efficiency, in vitro drug release, cellular uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl polyethylene glycol succinate nanoparticles. Biomaterials 2006;27:4025e33. [29] Gao X, Wang T, Wu B, Chen J, Chen J, Yue Y, et al. Quantum dots for tracking cellular transport of lectin-functionalized nanoparticles. Biochem Biophys Res Commun 2008;377:35e40.

[30] Kim S, Gaber MW, Zawaski JA, Zhang F, Richardson M, Zhang XA, et al. The inhibition of glioma growth in vitro and in vivo by a chitosan/ellagic acid composite biomaterial. Biomaterials 2009;30:4743e51. [31] Kievit FM, Florczyk SJ, Leung MC, Veiseh O, Park JO, Disis ML, et al. Chitosanalginate 3D scaffolds as a mimic of the glioma tumor microenvironment. Biomaterials 2010;31:5903e10. [32] Zhang XY, Chen J, Zheng YF, Gao XL, Kang Y, Liu JC, et al. Follicle-stimulating hormone peptide can facilitate paclitaxel nanoparticles to target ovarian carcinoma in vivo. Cancer Res 2009;69:6506e14. [33] Liang B, He ML, Chan CY, Chen YC, Li XP, Li Y, et al. The use of folate-PEGgrafted-hybranched-PEI nonviral vector for the inhibition of glioma growth in the rat. Biomaterials 2009;30:4014e20. [34] Beljebbar A, Dukic S, Amharref N, Bellefqih S, Manfait M. Monitoring of biochemical changes through the c6 gliomas progression and invasion by Fourier transform infrared (FTIR) imaging. Anal Chem 2009;81:9247e56. [35] Danhier F, Lecouturier N, Vroman B, Jerome C, Marchand-Brynaert J, Feron O, et al. Paclitaxel-loaded PEGylated PLGA-based nanoparticles: in vitro and in vivo evaluation. J Control Release 2009;133:11e7. [36] Zhang SQ, Song YN, He XH, Zhong BH, Zhang ZQ. Liquid chromatographytandem mass spectrometry for the determination of paclitaxel in rat plasma after intravenous administration of poly(L-glutamic acid)-alanine-paclitaxel conjugate. J Pharm Biomed Anal 2010;51:1169e74. [37] Petri B, Bootz A, Khalansky A, Hekmatara T, Muller R, Uhl R, et al. Chemotherapy of brain tumour using doxorubicin bound to surfactant-coated poly(butyl cyanoacrylate) nanoparticles: revisiting the role of surfactants. J Control Release 2007;117:51e8. [38] Hadjipanayis CG, Machaidze R, Kaluzova M, Wang L, Schuette AJ, Chen H, et al. EGFRvIII antibody-conjugated iron oxide nanoparticles for magnetic resonance imaging-guided convection-enhanced delivery and targeted therapy of glioblastoma. Cancer Res 2010;70:6303e12. [39] Farokhzad OC, Cheng J, Teply BA, Sherifi I, Jon S, Kantoff PW, et al. Targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo. Proc Natl Acad Sci U S A 2006;103:6315e20. [40] Perrault SD, Walkey C, Jennings T, Fischer HC, Chan WC. Mediating tumor targeting efficiency of nanoparticles through design. Nano Lett 2009;9: 1909e15. [41] Liu D, Mori A, Huang L. Role of liposome size and RES blockade in controlling biodistribution and tumor uptake of GM1-containing liposomes. Biochim Biophys Acta 1992;1104:95e101. [42] Danhier F, Vroman B, Lecouturier N, Crokart N, Pourcelle V, Freichels H, et al. Targeting of tumor endothelium by RGD-grafted PLGA-nanoparticles loaded with paclitaxel. J Control Release 2009;140:166e73. [43] Soundararajan S, Wang L, Sridharan V, Chen W, Courtenay-Luck N, Jones D, et al. Plasma membrane nucleolin is a receptor for the anticancer aptamer AS1411 in MV4-11 leukemia cells. Mol Pharmacol 2009;76:984e91. [44] Xie J, Wang CH. Electrospun micro- and nanofibers for sustained delivery of paclitaxel to treat C6 glioma in vitro. Pharm Res 2006;23:1817e26. [45] Chen DB, Yang TZ, Lu WL, Zhang Q. In vitro and in vivo study of two types of long-circulating solid lipid nanoparticles containing paclitaxel. Chem Pharm Bull (Tokyo) 2001;49:1444e7. [46] Xu Z, Gu W, Huang J, Sui H, Zhou Z, Yang Y, et al. In vitro and in vivo evaluation of actively targetable nanoparticles for paclitaxel delivery. Int J Pharm 2005;288:361e8. [47] Yu DH, Lu Q, Xie J, Fang C, Chen HZ. Peptide-conjugated biodegradable nanoparticles as a carrier to target paclitaxel to tumor neovasculature. Biomaterials 2010;31:2278e92. [48] Panagi Z, Beletsi A, Evangelatos G, Livaniou E, Ithakissios DS, Avgoustakis K. Effect of dose on the biodistribution and pharmacokinetics of PLGA and PLGAmPEG nanoparticles. Int J Pharm 2001;221:143e52. [49] Gu F, Zhang L, Teply BA, Mann N, Wang A, Radovic-Moreno AF, et al. Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers. Proc Natl Acad Sci U S A 2008;105:2586e91. [50] Kelland LR. Of mice and men: values and liabilities of the athymic nude mouse model in anticancer drug development. Eur J Cancer 2004;40:827e36. [51] van Weerden WM, Romijn JC. Use of nude mouse xenograft models in prostate cancer research. Prostate 2000;43:263e71. [52] Gao J, Jia WD, Li JS, Wang W, Xu GL, Ma JL, et al. Combined inhibitory effects of celecoxib and fluvastatin on the growth of human hepatocellular carcinoma xenografts in nude mice. J Int Med Res 2010;38:1413e27. [53] Gao ZW, Zhang DL, Guo CB. Paclitaxel efficacy is increased by parthenolide via nuclear factor-kappaB pathways in in vitro and in vivo human non-small cell lung cancer models. Curr Cancer Drug Targets 2010;10:705e15. [54] Ke XY, Zhao BJ, Zhao X, Wang Y, Huang Y, Chen XM, et al. The therapeutic efficacy of conjugated linoleic acid e paclitaxel on glioma in the rat. Biomaterials 2010;31:5855e64. [55] Xin H, Chen L, Gu J, Ren X, Wei Z, Luo J, et al. Enhanced anti-glioblastoma efficacy by PTX-loaded PEGylated poly(varepsilon-caprolactone) nanoparticles: in vitro and in vivo evaluation. Int J Pharm 2010;402:238e47. [56] Hobbs SK, Monsky WL, Yuan F, Roberts WG, Griffith L, Torchilin VP, et al. Regulation of transport pathways in tumor vessels: role of tumor type and microenvironment. Proc Natl Acad Sci U S A 1998;95:4607e12.