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Cancer Research 66, 11878, December 15, 2006. doi: 10.1158/0008-5472.CAN-06-2354
© 2006 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Cationic Albumin–Conjugated Pegylated Nanoparticles Allow Gene Delivery into Brain Tumors via Intravenous Administration

Wei Lu1, Qing Sun2, Jin Wan3, Zhenjue She3 and Xin-Guo Jiang1

1 Department of Pharmaceutics, School of Pharmacy; 2 Gene Research Center; and 3 Department of Anatomy, Histology, and Embryology, Shanghai Medical School, Fudan University, Shanghai, P.R. China

Requests for reprints: Xin-Guo Jiang, Department of Pharmaceutics, School of Pharmacy, Fudan University (Fenglin Campus), P.O. Box 130, 138 Yi Xue Yuan Road, Shanghai 200032, P.R. China. Phone: 86-21-5423-7381; Fax: 86-21-5423-7381; E-mail: xgjiang{at}shmu.edu.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Patients with malignant gliomas have a poor prognosis because these tumors do not respond well to conventional treatments. Studies of glioma xenografts suggest that they may be amenable to gene therapy with cytotoxic genes, such as the proapoptotic Apo2 ligand/tumor necrosis factor–related apoptosis-inducing ligand (Apo2L/TRAIL). Gene therapy of gliomas ideally employs i.v. given vectors, thus excluding viral vectors as they cannot cross the brain microvascular endothelium or blood-brain barrier. Recently, we reported the synthesis of cationic albumin–conjugated pegylated nanoparticles (CBSA-NP) and showed their accumulation in mouse brain cells upon i.v. administration. In this study, plasmid pORF-hTRAIL (pDNA) was incorporated into CBSA-NP, and the resulting CBSA-NP-hTRAIL was evaluated as a nonviral vector for gene therapy of gliomas. Thirty minutes after transfection of C6 glioma cells, CBSA-NP-hTRAIL was internalized and mostly located in the cytoplasm, whereas NP-hTRAIL was entrapped in the endolysosomal compartment. At 6 and 48 hours after transfection, respectively, released pDNA was present in the nuclei and induced apoptosis. At 30 minutes after i.v. administration of CBSA-NP-hTRAIL to BALB/c mice bearing i.c. C6 gliomas, CBSA-NP-hTRAIL colocalized with glycoproteins in brain and tumor microvasculature and, via absorptive-mediated transcytosis, accumulated in tumor cells. At 24 and 48 hours after i.v. administration of CBSA-NP-hTRAIL, respectively, hTRAIL mRNA and protein were detected in normal brain and tumors. Furthermore, repeated i.v. injections of CBSA-NP-hTRAIL induced apoptosis in vivo and significantly delayed tumor growth. In summary, this study indicates that CBSA-NP-hTRAIL is a promising candidate for noninvasive gene therapy of malignant glioma. (Cancer Res 2006; 66(24): 11878-87)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Patients with malignant glioma have a poor prognosis because these brain tumors respond poorly to radiation or chemotherapy, the conventional treatments of cancer (1). Instead, gene therapy with cytotoxic genes, such as the proapoptotic Apo2 ligand/tumor necrosis factor–related apoptosis-inducing ligand (Apo2L/TRAIL), may offer therapeutic promise. Most glioma cell lines express the agonist Apo2L/TRAIL receptors but no or undetectable levels of the antagonist receptors (2). Normal cells, on the other hand, have been found to express antagonist Apo2L/TRAIL receptors (3, 4). Thus, Apo2L/TRAIL may allow selective killing of tumor cells only. I.t. administration of Apo2L/TRAIL has induced tumor regression in a xenograft model with i.c. glioma implantation (5, 6).

Yet, the brain capillary endothelium, which with its tight junctions forms the brain-blood barrier (BBB) in vivo, poses a major obstacle for delivery of exogenous genes into brain tumors (7). Viral vectors, which have been used for efficient delivery of exogenous genes into peripheral tumors, cannot cross the BBB, and their in vivo delivery into brain tumors requires highly invasive administration routes (7–9). This makes multi-dosing difficult, while the viral vectors themselves are associated with immunogenicity, inflammation, oncogenic properties, and unknown long-term effects in patients (7, 10, 11).

The difficulties with the viral vectors have led to the development of nonviral vectors, such as pegylated immunoliposomes, which allow gene delivery into the brain via i.v. administration (12–14). Targeting of the pegylated immunoliposomes to the BBB is attained by monoclonal antibodies against either the transferrin receptor or insulin receptor, which upon binding to their ligands trigger receptor-mediated endocytosis (15). Upon i.v. administration of pegylated immunoliposomes, expression of a pegylated immunoliposome–encapsulated gene has been shown in human glioma cells implanted i.c. in mice (13).

Pegylated nanoparticles, compared with liposomes, are physically and chemically more stable and allow lyophilization for long-term storage (16, 17). Cationic albumin–conjugated pegylated nanoparticles (CBSA-NP), recently developed in our laboratory, have shown brain delivery property (18, 19). A higher accumulation of CBSA-NP than of native albumin–conjugated pegylated nanoparticles has been found in mouse brain cells after i.v. administration of these nanoparticles loaded with a fluorescent probe (19). Free CBSA has been reported to target delivery to the BBB in brain capillaries via absorptive-mediated transcytosis (AMT; refs. 20, 21).

To determine the potential of CBSA-NP as a nonviral vector for the systemic delivery of exogenous genes into brain, we chose hTRAIL-encoding plasmid (pORF-hTRAIL) as a model therapeutic gene for malignant glioma. The mechanisms of gene delivery by CBSA-NP-hTRAIL into normal brain and brain tumor and the intracellular fate of this nanoparticle were studied. Expression of hTRAIL in tumors and its antitumor effect were evaluated in C6 glioma tumors i.c. implanted in BALB/c mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and animals. CBSA and copolymers of MPEG-PLA (molecular weight = 43 kDa) and maleimide-PEG-PLA (molecular weight = 44 kDa) were synthesized in our laboratory, as previously described (19). Plasmid pORF-hTRAIL (v.16, 4,058 bp) was purchased from Invivogen (San Diego, CA). Fetal bovine serum (FBS) was from Life Technologies/Invitrogen (Carlsbad, CA). 6-Coumarin was from Sigma-Aldrich (St. Louis, MO). Texas Red-transferrin, LysoTracker Blue, ethidium monoazide (EMA), Alexa Fluor 633-wheat germ agglutinin (WGA), CellTracker CM-DiI, and PicoGreen DNA quantitative analysis kit were from Molecular Probes (Eugene, OR). Rabbit anti-human/mouse/rat active caspase-3 polyclonal antibody was from Chemicon (Temecula, CA). hTRAIL ELISA kit was from Biosource (Camarillo, CA). LipofectAMINE 2000 was from Invitrogen (Carlsbad, CA). Goat anti-human TRAIL-specific IgG and in situ apoptosis detection kit (TACS TdT kit) were from R&D Systems (Minneapolis, MN). DNA ladder was from MBI (Fermentas International, Canada). Rabbit anti-goat IgG-Cy3 antibody, Heparin-biotin sodium salt, streptavidin-gold (10-nm colloidal gold), and 4,6-diamidino-2-phenylindole (DAPI) were from Sigma-Aldrich. Poly(ethyleneimine) (molecular weight = 1,800 Da) was from Polysciences, Inc. (Warrington, PA). Double-distilled water was purified using a Millipore Simplicity System (Millipore, Bedford, MA). All other chemicals were analytic reagent grades and used without further purification. The rat C6 glioma cell line (CCL-107) was obtained from the American Type Culture Collection (Rockville, MD). The BALB/c mice (Department of Experimental Animals, Fudan University, Shanghai, China) used in this study were treated according to protocols approved by the ethical committee of Fudan University.

Preparation of nanoparticles loaded with plasmid DNA. pORF-hTRAIL (hereafter pDNA) was amplified in the Escherichia coli Top10, extracted by alkaline lysis, and purified using the plasmid Giga column isolation kit (Qiagen GmbH, Hilden, Germany). To prepare CBSA-NP-hTRAIL, pDNA and thiol-reactive pegylated nanoparticles were subjected to a double emulsion and solvent evaporation technique according to our previous method (19). Hereto, the internal water phase included pDNA (250 µg) in 50 µL of Tris-EDTA buffer (pH 7.5). Oil phase was 1 mL of dichloromethane solution containing MPEG-PLA (24.0 mg) and maleimide-PEG-PLA (2.4 mg). The sonication procedures of primary and secondary emulsion were set as 120 W, 15 seconds (continuous) and 120 W, 15 seconds (intermittent), respectively. After evaporation and removal of unencapsulated pDNA by washing, the thiol-reactive pegylated nanoparticles were slowly mixed with thiolated CBSA (19) at a 1:1 thiolated/maleimide ratio on a rotating plate for 8 hours. The resulting CBSA-NP-hTRAIL was purified through Sepharose CL-4B column eluted with saline. pORF-hTRAIL–loaded MPEG-PLA nanoparticle (NP-hTRAIL) was prepared similarly to CBSA-NP-hTRAIL, except that MPEG-PLA (26.4 mg/mL) was used only.

To allow analysis of their cellular uptake and intracellular distribution, CBSA-NP-hTRAIL and NP-hTRAIL were labeled with the fluorescent dye 6-coumarin, as previously described (22).

Furthermore, to allow for its intracellular tracking, pDNA was covalently labeled with the fluorescent dye EMA by photolysis, as described elsewhere (23). The preparation of 6-coumarin-labeled CBSA-NP containing EMA-labeled pDNA (CBSA-NP-hTRAIL-EMA) was similar to that of 6-coumarin-labeled CBSA-NP-hTRAIL, except that EMA-labeled pDNA was used instead of pDNA.

Nanoparticle characterization. Mean (volume based) diameter and zeta potential of the nanoparticles were determined by dynamic light scattering using a Zeta Potential/Particle Sizer Nicomp 380 ZLS (Particle Sizing Systems, Santa Barbara, CA). For zeta potential measurements, nanoparticle suspensions (200 µg/mL in 0.001 mol/L HEPES buffer) were pH adjusted with 0.1 mol/L sodium hydroxide or 0.1 mol/L hydrochloric acid. Nanoparticles were visualized with scanning (JOEL 6320FV, Philips/FEI, Hillsboro, OR) and transmission (JOEL CM1200, Philips/FEI) electron microscopes. Drug encapsulating and loading efficiencies of CBSA-NP-hTRAIL and NP-hTRAIL were determined, as previously described (19). Covalent conjugations between CBSA and nanoparticles were confirmed by immunostaining of CBSA-NP-hTRAIL with heparin-biotin and streptavidin-gold complexes (19).

The ability of CBSA to protonate and obtain a positive charge over a 10 to 2 pH range was determined by acid-base titration (24, 25). Briefly, 10 mg CBSA, native bovine serum albumin (BSA), or poly(ethyleneimine) was dissolved in 10 mL of 150 mmol/L NaCl, adjusting to pH 10 with 1 mol/L NaOH. The pH of the solution was then monitored as aliquots of 0.1 mol/L HCl were added.

The release of pDNA from both CBSA-NP-hTRAIL and NP-hTRAIL were done in Tris-EDTA buffer (26). The amount of plasmid released in each time interval was determined by the PicoGreen assay, which is very sensitive for supercoiled and double-strained DNA, but not for single-strained DNA and oligonucleotides (17). pDNA stability was determined by gel electrophoresis (1% agarose containing ethidium bromide, 110 V, 90 minutes).

Cellular uptake of CBSA-NP-hTRAIL and NP-hTRAIL. CBSA-NP-hTRAIL and NP-hTRAIL were labeled with the lipophilic fluorescent dye 6-coumarin (~0.1% loading) to allow analysis of their cellular uptake and intracellular fate (22). C6 cells (16 x 103 per 200 µL/well) were seeded in 96-well plates in RPMI 1640, 10% FBS. After 24 hours and a 30-minute preincubation in RPMI 1640, cells were treated with 100 µg/mL of 6-coumarin-labeled CBSA-NP-hTRAIL or NP-hTRAIL and various inhibitors for 30 minutes: RPMI 1640 (control), CBSA (0.1 or 1 mg/mL), polylysine (50 or 500 µg/mL), 0.1% w/v sodium azide, 450 mmol/L sucrose, 20 µmol/L phenylarsinoxide, or 10 µg/mL filipin. Cells were washed with ice-cold PBS, acid buffer [120 mmol/L NaCl, 20 mmol/L sodium barbital, 20 mmol/L sodium acetate (pH 3)] at 4°C for 5 minutes and ice-cold PBS. Each well received 50 µL PBS and 150 µL DMSO, and 6-coumarin fluorescence was measured by spectrofluorometry. A standard curve was constructed by measuring samples of 150 µL DMSO and 50 µL PBS with increasing concentrations of the nanoparticles. Samples were tested in pentaplicate. Nanoparticle uptake was presented as percent uptake of the control.

Intracellular tracking of nanoparticles and pDNA. C6 cells (1 x 104 per coverslip) were plated on glass coverslips coated with L-polylysine. After 24 hours and a 30-minute preincubation in RPMI 1640, cells were treated for 30 minutes with a suspension of 6-coumarin-labeled CBSA-NP-hTRAIL or NP-hTRAIL (100 µg/mL) in RPMI 1640 with markers of endolysosomal compartments, LysoTracker Blue (50 nmol/L), and of early and recycling endosomes, Texas Red-transferrin (100 µg/mL). Cells were washed, fixed, and mounted in DakoCytomation fluorescent mounting medium. Images were captured with UV (LysoTracker Blue), fluorescein (6-coumarin), and rhodamine (Texas Red-transferrin) filters using a Zeiss dual photon LSM 510 confocal microscope. Images were superimposed to determine the intracellular nanoparticle localization.

To track pDNA intracellularly, it was covalently labeled with the fluorescent dye EMA. C6 cells plated as above and preincubated with RPMI 1640 for 30 minutes were treated with CBSA-NP-hTRAIL-EMA (100 µg/mL) for 30 minutes. Cells were then incubated with blank RPMI 1640 and after 5 hours were treated with LysoTracker Blue (50 nmol/L) for 30 minutes. After washing, fixing, and mounting steps, slides were examined by confocal microscopy, using a rhodamine filter for EMA-labeled DNA.

In vitro gene expression and cell apoptosis determination. C6 cells (1 x 105 per well) were cultured in six-well plates until 70% confluency was reached. Cells were incubated with RPMI 1640 for 15 minutes and transfected with 2 mL serum-free medium containing LipofectAMINE 2000 plus 4 µg pDNA, CBSA-NP-hTRAIL with 10 µg encapsulated pDNA, or NP-hTRAIL with 10 µg encapsulated pDNA (controls: medium, CBSA-NP, or NP). After 5 hours, medium was replaced by 2 mL complete medium. Forty-eight hours after transfection, soluble hTRAIL in the supernatant was measured using a hTRAIL ELISA kit. Cells were trypsinized, stained by propidium iodide, and analyzed by fluorescence-activated cell sorter for apoptotic cells (FACSCalibur flow cytometry system, BD Biosciences, San Jose, CA).

I.c. tumor implantation. To allow in vivo tracking of proliferation, C6 cells were labeled with the vital dye CellTracker CM-DiI, according to the manufacturer's protocol. C6 cells were not labeled for other in vivo studies. C6 cells (5 x 104 in 4 µL RPMI 1640) were implanted into the right striatum (2 mm lateral to the bregma and 3 mm of depth) of BALB/c mice by using a stereotactic fixation device with mouse adaptor (Benchmark; ref. 6).

In vivo tracking of nanoparticles. Seven days after implantation, mice received 6-coumarin-labeled CBSA-NP-hTRAIL (control: 6-coumarin-labeled NP-hTRAIL) via the tail vein at a dose of 60 mg/kg body weight. After 30 minutes, animals were sacrificed, and brain coronal cryostat sections (5 µm) were prepared and viewed with a confocal microscope using rhodamine (CellTracker CM-DiI) and fluorescein (6-coumarin) filters. WGA specifically binds negatively charged endothelial microdomains (27). To detect glycoproteins, sections were stained with Alexa Fluor 633-WGA (2.5 µg/mL, room temperature, 1 hour) and viewed with a far-red filter. For intracellular tracking of nanoparticles, images were taken along the z-axis at 0.1-µm intervals, and three-dimensional images were reconstructed using Carl Zeiss AIM software (version 3.2).

CBSA-NP loaded with osmium tetroxide was prepared by adding 1 mg osmium tetroxide to the polymer solution before emulsification. At 7 days after implantation, mice received CBSA-NP-osmium tetroxide via the tail vein. Thirty minutes thereafter, brain tumors were removed and prepared for examination by a Philips CM120 transmission electron microscope, as previously described (28).

Gene expression in vivo. At day 7 after implantation, mice received CBSA-NP-hTRAIL, NP-hTRAIL, or saline (control) via the tail vein at a dose of 100 µg pDNA/kg body weight. Mice were sacrificed after 24 hours for in situ hybridization (ISH) or after 48 hours for immunohistochemistry. For ISH, 5-µm brain coronal cryostat sections were prepared after perfusing anesthesized mice with 4% paraformaldehyde. ISH used a custom-synthesized biotin-labeled hTRAIL-specific oligonucleotide probe (5'-bio-ttgccagcaggggctgttcatactctcttcgtc-3'; Shenergy Biocolor BioScience and Technology Co., Shanghai, China) and the DNADetector Chromogenic In situ Hybridization kit (KPL, Gaithersburg, MD). After proteinase K digestion, tissue was hybridized with 50% formamide hybridization cocktail (equal volumes of 2 x hybridization buffer and formamide) containing biotin-labeled probe (1 ng/mL, 37°C, 18 hours) and developed with TrueBlue. No endogenous mTRAIL expression was detected upon examination by light microscopy.

Immunohistochemical detection of hTRAIL protein used goat anti-hTRAIL IgG (1:100) as primary antibody followed by Cy3-labeled secondary antibodies (1:50). After DAPI counterstaining, slides were mounted and examined by fluorescence microscopy (Olympus, Tokyo, Japan).

To detect tumor hTRAIL expression-time course, tumor tissues of post-implantation mice were collected at 0.5, 1, 2, 4, and 6 days after dosing. Whole protein extracts were prepared by direct addition of cold lysis buffer to the samples and mechanical homogenization of tissue (29). Protein quantitation was done using bicinchoninic acid protein assay kit (Shenergy Biocolor BioScience and Technology) and 50 µg of protein was loaded on 10% SDS-PAGE gels. Western blot analysis of hTRAIL was according to previous described method (29). The dilution of primary antibody was 1:1,000. Horseradish peroxidase–conjugated rabbit anti-IgG goat (dilution 1:2,000; Pierce Biotech, Inc., Rockford, IL) was detected by chemiluminescence using Super Signal West Femto Maximum Sensitivity Substrate (Pierce Biotech).

Immunohistochemistry of apoptosis markers and survival monitoring. At days 8, 10, and 12 after implantation, mice received CBSA-NP-hTRAIL, NP-hTRAIL, or saline (control) via the tail vein at a dose of 100 µg pDNA/kg body weight (1-week treatment, three doses per week). At day 14 after implantation, mice were sacrificed, except for 10 mice of each treatment group, which were monitored for survival. The sacrificed mice were used to prepare brain coronal paraffin sections (5 µm). Slides were either immunostained with anti-active caspase-3 (1:100) or subjected to terminal deoxynucleotidyl transferase–mediated nick-end-labeling (TUNEL; TACS TdT kit) to detect nuclear DNA fragments. Slides were developed with diaminobenzidine and counterstained with methyl green. Besides the above 1-week treatment groups, an extended treatment group with the same dose of CBSA-NP-hTRAIL at days 8, 10, 12, 15, 17, and 19 after implantation (2-week treatment, three doses per week) was conducted for survival monitoring.

Statistical analysis. Differences between treatment groups in the in vitro cellular uptake and protein expression assays were assessed using an unpaired Student's t test. Survival data were presented using Kaplan-Meier plots and were analyzed using a log-rank test. P < 0.05 was considered significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of nanoparticles. No significant differences in particle size were found between NP-hTRAIL (108.6 ± 10.8 nm) and CBSA-NP-hTRAIL (115.7 ± 13.9 nm), suggesting that CBSA conjugation did not affect particle size. No differences were observed either in drug encapsulating efficiency (65.0 ± 6.1% versus 57.5 ± 8.3%) or drug loading efficiency (0.52 ± 0.05% versus 0.46 ± 0.07%). Scanning electron micrography and transmission electron micrography (TEM) showed that CBSA-NP-hTRAIL were spherical and of equal size (Fig. 1A1 and A2 ). Following a two-step immunostaining process using heparin/biotin and streptavidin/gold complexes, TEM detected gold particles on the surface of CBSA-NP-hTRAIL, indicative of covalent conjugations between CBSA and nanoparticle (Fig. 1A3). The average zeta potential of CBSA-NP-hTRAIL was –15.4 ± 1.3 mV at pH 7, but increased with decreasing pH, reaching 11.3 ± 1.8 mV at pH 2 (Fig. 1B1). In contrast, the zeta potential of NP-hTRAIL changed little and remained negative with decreasing pH (–18.1 ± 0.9 mV at pH 7 and –11.3 ± 0.9 mV at pH 2).


Figure 1
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  		Figure 1. Characterization of CSBA-NP-hTRAIL and the pDNA (pORF-hTRAIL) encapsulated within CBSA-NP. A1, scanning electron micrograph of CBSA-NP-hTRAIL (x20K; 30 kV; bar, 200 nm). TEMs of (A2) CBSA-NP-hTRAIL and (A3) CBSA-NP-hTRAIL in which CBSA is labeled with 10-nm colloidal gold after a two-step immunostaining process using heparin/biotin and streptavidin/gold complexes (1% phosphotungstic acid–negative staining; x120K; bar, 50 nm.). B1, zeta potentials of nanoparticles (200 µg/mL) in 0.001 mol/L HEPES buffer adjusted to different pH values with either 0.1 mol/L sodium hydroxide or 0.1 mol/L hydrochloric acid. Points, mean (n = 5); bars, SD. Note that CBSA-NP-hTRAIL but not NP-hTRAIL is cationic at acidic pH values. B2, acid-base titration: 10 mg CBSA, BSA, or PEI1800 was dissolved in 10 mL of 150 mmol/L NaCl, adjusted to pH 10 with 1 mol/L NaOH. The solution was titrated with increasing volume of 0.1 mol/L HCl. C1, cumulative release (% amount loaded) of pDNA from pDNA-loaded nanoparticles. Release was studied in Tris-EDTA buffer (pH 7.4) at 37°C. Points, mean (n = 4); bars, SD. C2, analysis of pDNA (pORF-hTRAIL) stability by agarose gel electrophoresis. Lane 1, DNA ladder; lane 2, original pDNA; lanes 3 and 6, pDNA extracted from CBSA-NP-hTRAIL and NP-hTRAIL, respectively; lanes 4 and 7, pDNA released from CBSA-NP-hTRAIL and NP-hTRAIL after 1 day, respectively; lanes 5 and 8, pDNA released from CBSA-NP-hTRAIL and NP-hTRAIL after 10 days, respectively. In lanes 2 to 8, the top strands are the relaxed conformation of pDNA, whereas the bottom strands are the supercoiled conformation.

 
As shown in Fig. 1B2, poly(ethyleneimine) has a strong proton-buffering effect at pH range from 10 to 4, which was attributed to protonation of the primary amine groups (pH >9) and tertiary and secondary amines (pH range, 9–4; ref. 30). However, CBSA is a weaker pH buffering system. The charge contribution of CBSA included primary amine group introduced by ethylenediamine following CBSA synthesis (19) and carboxyl group of acidic amino acid residues. The former caused the initial high pH protonation (pH >9), whereas the cationization of CBSA at pH 9 to 4 might be attributed to protonation of carboxyl groups. Compared with CBSA, BSA has no proton-buffering capacity at pH >7 but a weak protonation at pH 7 to 4.

Both of the particles exhibited a similar biphasic pDNA release pattern in Tris-EDTA buffer (pH 7.4), which was characterized by a first initial rapid release (>50% of pDNA was released within the first day) followed by a slower and continuous release (80% was released in 10 days; Fig. 1C1). Electrophoresis result shows the ratio between relaxed to supercoiled conformation of pDNA extracted and 1-day released from CBSA-NP-hTRAIL in comparison with control pDNA increased from 10:90% to 60:40% (Fig. 1C2). The relaxed/supercoiled ratio of pDNA increased to 90:10% after a 10-day release. However, no DNA fragments were detected, which has been observed before and hypothesized to reflect a protective effect of the amphiphilic PLA-PEG on shearing of pDNA (17, 26). Analysis of pDNA extracted or released from NP-hTRAIL shows a similar pattern as those from CBSA-NP-hTRAIL.

CBSA-NP-hTRAIL and NP-hTRAIL uptake. The uptake of both CBSA-NP-hTRAIL and NP-hTRAIL by C6 cells was energy dependent, as their uptake was reduced to 72.9% and 66.9%, respectively, after energy depletion by sodium azide (Fig. 2A ). Phenylarsinoxide and hypertonic sucrose, inhibitors of endocytosis, also significantly decreased the intracellular uptake of CBSA-NP-hTRAIL and NP-hTRAIL. However, filipin, a specific inhibitor of caveolae-associated endocytosis, decreased uptake of CBSA-NP-hTRAIL but not NP-hTRAIL. Furthermore, free polycations, such as CBSA and polylysine, dose-dependently inhibited uptake of CBSA-NP-hTRAIL, but high doses of these polycations did not inhibit uptake of NP-hTRAIL, suggesting that CBSA-NP-hTRAIL is subject to AMT.


Figure 2
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  		Figure 2. Cellular and organelle uptake and cytotoxic activity of CBSA-NP-hTRAIL and NP-hTRAIL. A, uptake of CBSA-NP-hTRAIL and NP-hTRAIL by C6 cells in the presence of various inhibitors of transcytosis. Uptake is shown as percentage of uptake by control cells. **, P < 0.01, unpaired Student's t test. Columns, mean of five independent experiments; bars, SD. PheAsO, phenylarsinoxide. B, intracellular tracking of nanoparticles and pDNA incorporated into nanoparticles. B1 to B2, C6 cells were incubated with LysoTracker Blue to label secondary endosomes and lysosomes, Texas red-transferrin to stain early and recycling endosomes, and 6-coumarin-labeled CBSA-NP-hTRAIL or NP-hTRAIL (green fluorescence). Thirty minutes after incubation, C6 cells were analyzed by confocal microscopy. B1, 6-coumarin-labeled CBSA-NP-hTRAIL (green) was predominantly detected in the cytoplasm and outside the endolysosomal compartment (blue and red), indicating its rapid uptake by C6 cells and its fast endolysosomal escape. B2, detection of 6-coumarin-labeled NP-hTRAIL (green) showed its entrapment in the secondary endosomes and lysosomes (blue), not in the early and recycling endosomes (red). B3, to enable its intracellular tracking, pDNA (pORF-hTRAIL) was covalently labeled with the fluorescent dye EMA. C6 cells were treated with 6-coumarin-labeled CBSA-NP-hTRAIL-EMA for 30 minutes. Cells were washed, incubated in blank medium for 5 hours, treated with LysoTracker Blue for 30 minutes, and analyzed by confocal microscopy. After 6 hours, some pDNA remained encapsulated (yellow), and some pDNA had released (red) from CBSA-NP and had reached nuclei (arrows). CBSA-NP (green) was present in the cytoplasm. Bar, 10 µm. C, fluorescence-activated cell sorting analysis of C6 cells 48 hours after transfection with different vectors. Apoptosis was observed only after transfection of pORF-hTRAIL incorporated into CBSA-NP or LipofectAMINE.

 
Endolysosomal escape of nanoparticles and intracellular sorting of pDNA. To track the fate of the nanoparticles intracellularly, C6 cells were incubated with Texas Red-transferrin (red fluorescent marker for early and recycling endosomes), LysoTracker Blue (blue fluorescent marker for secondary endosomes and lysosomes), and the 6-coumarin-labeled nanoparticles. At 30 minutes after incubation, the superimposed image of Fig. 2B1 shows that green fluorescence was both inside and outside the red and blue compartments, indicating that CBSA-NP-hTRAIL were not only inside endosomes and lysosomes but had spread throughout the cytoplasm. This suggested that following their uptake CBSA-NP-hTRAIL escaped rapidly from the endolysosomal compartments to the cytoplasm. Unlike CBSA-NP-hTRAIL, almost all green fluorescence of NP-TRAIL overlapped with blue fluorescence, indicating NP-hTRAIL was entrapped in the secondary endosome and lysosome rather than the early and recycling endosomes (Fig. 2B2, superimposed image). The absence of green fluorescence from the cytoplasm suggested a different intracellular sorting mechanism for NP-hTRAIL.

In gene therapy, encapsulated DNA should translocate to the nucleus once released inside the cytoplasm. To allow for separate tracking of the encapsulated pDNA and CBSA-NP, pDNA was covalently bound to the red fluorescent marker EMA (23), whereas CBSA-NP was labeled with 6-coumarin. As depicted in the superimposed image of Fig. 2B3, an intense red fluorescent signal was found inside rounded and well-defined structures (i.e., nuclei, at 6 hours after incubation). The predominantly red, not yellow, nuclear fluorescence indicated that CBSA-NP and pDNA separated some time before the latter entered the nucleus. Such rapid dissociation may be attributed to initial rapid release kinetics, which about 40% of pDNA was released from CBSA-NP within the first 4 hours (Fig. 1C1). pDNA encapsulated inside CBSA-NP seemed to enter the nucleus much faster than the plasmid encapsulated inside OX26-coupled pegylated liposomes, which entered the nucleus 24 hours after receptor-mediated endocytosis (13).

In vitro transfection. Expression of supernatant hTRAIL protein by C6 cells at 24 hours after transfection illustrated the high transfection efficiency mediated by CBSA-NP. With 2.5-fold the amount of DNA in LipofectAMINE 2000, CBSA-NP-hTRAIL induced a hTRAIL protein level similar to that of LipofectAMINE 2000/hTRAIL (1,192 ± 55 pg/mL versus 1,133 ± 44 pg/mL) and significantly higher than that of NP-hTRAIL (470 ± 30 pg/mL). Levels of hTRAIL protein after transfection with blank CBSA-NP, NP, or vehicle-free culture medium were below the ELISA detection limit (46.8 pg/mL). As shown by fluorescence-activated cell sorting analysis, the apoptotic effect of CBSA-NP-hTRAIL, with 2.5-fold the amount of DNA in LipofectAMINE 2000, was similar to that of LipofectAMINE 2000/hTRAIL and about five times higher than that of NP-hTRAIL (Fig. 2C). Neither blank CBSA-NP nor NP caused apoptosis, showing the low toxicity of these nanoparticulate vectors.

Targeted transportation of CBSA-NP inside tumor. To distinguish between donor and recipient cells after i.c. transplantation, C6 cells were labeled with the red fluorescent, lipophilic tracer CM-DiI. CM-DiI-labeled C6 cells exhibited minimal cytotoxicity with cellular fluorescence labeling efficiency over 95% (Fig. 3A1 ). Seven days after i.c. transplantation of the CM-DiI–labeled C6 cells, microscopic examination showed that they had good cell vitality and had grown into distinct tumor tissue displaying red fluorescent cyto-lipid separation (Fig. 3A2). Thirty minutes after i.v. administration, more green fluorescence of CSBA-NP-hTRAIL than of NP-hTRAIL was detected in the tumor region, indicative of the brain tumor–specific accumulation of CBSA-NP-hTRAIL (Fig. 3A2). Three-dimensional reconstruction of brain tumor images through x-, y-, and z-axes showed colocalization of red and green fluorescence, suggesting that CBSA-NP-hTRAIL was taken in by tumor cells rather than remaining stagnant in the tumor interstitium (Fig. 3A3).


Figure 3
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  		Figure 3. In vivo uptake of CBSA-NP-hTRAIL and NP-hTRAIL into normal brain tissue and i.c. implanted C6 brain tumor. C6 cells were labeled with the vital dye CellTracker CM-DiI and were i.c. implanted into the right striatum of BALB/c mice. Seven days after implantation, 6-coumarin-labeled CBSA-NP-hTRAIL or NP-hTRAIL was injected i.v., and after 30 minutes, the mice were sacrificed. Coronal cryostat sections of normal brain and brain tumor tissue were analyzed by confocal microscopy. A, i.t. accumulation of CBSA-NP-hTRAIL and NP-hTRAIL. A1, C6 cells labeled with CM-DiI (red) before i.c. implantation. A2, CBSA-NP-hTRAIL (green) accumulated more in brain tumor tissue (red) than NP-hTRAIL (green). A3, three-dimensional reconstruction of brain tumor images showed CBSA-NP-hTRAIL (green) to be inside brain tumor cells (red). Bar, 50 µm (A1–A2) and 5 µm (A3). B, colocalization of CBSA-NP-hTRAIL and NP-hTRAIL with glycoproteins in brain vasculature and brain tumors. Coronal cryostat sections of normal brain and brain tumor tissue were stained with Alexa Fluor 633-WGA, which specifically binds to negatively charged residues in endothelium, and analyzed by confocal microscopy. B1 to B2, in normal brain, WGA-stained glycoproteins (purple, pseudocolor) were mainly distributed in the endothelial membrane. B3, at higher magnification, transcytosis of CBSA-NP-hTRAIL (green) across the BBB was shown by its binding to the luminal side (white arrowhead), its presence inside the cytoplasm (yellow arrowhead), and its binding to the abluminal side (red arrowhead) of brain capillary endothelial cells (purple membrane). B4, transcytosis (arrows) of NP-hTRAIL (green) in normal brain vasculature did not involve colocalization with glycoproteins (purple). B5 to B7, in brain tumors, the negatively charged residues (purple) were highly expressed in both tumor endothelial cells and tumor cells. Brain tumor accumulation of (B5) CBSA-NP-hTRAIL (green) was higher than that of (B6) NP-hTRAIL (green), with colocalization (white) of most of CBSA-NP-hTRAIL and the anionic residues. B7, three-dimensional reconstruction of brain tumor images shows several CBSA-NP-hTRAIL (green) specifically bound to the glycoproteins (purple) present in the tumor vascular wall. A, abluminal side of BBB; L, luminal side of BBB; V, blood vessel. Bar, 20 µm (B1–B7). C, absorptive-mediated transcytosis of CBSA-NP across brain tumor microvasculature into brain tumor. Mice carrying i.c. implanted C6 brain tumors were injected i.v. with CBSA-NP loaded with osmium tetroxide at day 7 after implantation. After 30 minutes, brain tumors were removed and prepared for analysis by TEM. C1, TEM of brain tumor microvasculature revealed a pore cutoff size <100 nm (arrowhead). C2, CBSA-NP was found in endocytic vesicles of tumor endothelial cells (arrow). C3, in tumor cells, CBSA-NP was detected interacting with the vesicle membrane (arrow) and free in the cytoplasm (arrowheads). *, blood vessel; {pi}, tumor interstitium; e, tumor endothelial cells; t, tumor cells. Bar, 200 nm (C1–C2) and 500 nm (C3).

 
To enter the brain via AMT, CBSA-NP-hTRAIL needs to selectively bind to negatively charged glycoproteins in the brain capillary wall. To elucidate this interaction in vivo, a far-red fluorescent WGA was used to detect such glycoproteins; negatively charged glycoproteins were located in brain vasculature rather than parenchyma (Fig. 3B1 to B2). Higher magnifications showed that they were expressed in both the luminal and abluminal membranes of brain endothelial cells (Fig. 3B3 to B4). As shown in Fig. 3B3, at 30 minutes after CBSA-NP-hTRAIL injection, the colocalization of fluorescent WGA with CBSA-NP-hTRAIL was detected not only in the luminal membrane of endothelial cells (white arrowhead) but also in the abluminal membrane (red arrowhead). CBSA-NP-hTRAIL was further being transported across the cytoplasm of endothelial cells (yellow arrowhead). This suggested that transcytosis of CBSA-NP-hTRAIL across the BBB might involve its electrostatic binding to the negatively charged glycoproteins in the endothelial cell membrane. The transcytosis of NP-hTRAIL, however, seemed to be different because NP-hTRAIL and glycoproteins in the endothelial cell membrane did not colocalize (Fig. 3B4, arrows).

In contrast to normal brain parenchyma, brain tumor expressed higher levels of negatively charged glycoproteins, not only in tumor vasculature but also in tumor cells (Fig. 3B5 to B6). CBSA-NP-hTRAIL was found to be far more extensively distributed in tumors than NP-hTRAIL. Moreover, white color in tumor tissue showed the strong colocalization of CBSA-NP-hTRAIL and negatively charged glycoproteins (Fig. 3B5). Three-dimensional reconstruction further showed that CSBA-NP-hTRAIL was overlapped with glycoproteins in the tumor vessel wall, and that its transport across tumor blood vessels involved the specific binding to anionic glycoproteins (Fig. 3B7). Virtually no colocalization between NP-hTRAIL and negatively charged glycoproteins was detected in brain tumor tissue (Fig. 3B6). Brain tumor–specific accumulation of CBSA-NP-hTRAIL was further substantiated by biodistribution data, which showed that increases in tissue clearance and uptake of CBSA-NP-hTRAIL were much higher in tumor than in normal brain tissues (see Supplementary Data).

TEM of brain tumor vasculature proved that its pore cutoff size was <100 nm (Fig. 3C1, arrowhead). CBSA-NP was clearly seen adhering to membrane of endocytic vesicles of tumor endothelial cells (Fig. 3C2) and was further detected in the endolysosomal compartment and cytoplasm of tumor cells (Fig. 3C3). These results further proved that accumulation of CBSA-NP in brain tumor tissue (Fig. 3A2) was accounted for by AMT.

hTRAIL gene expression and tumor apoptosis. At 24 hours after i.v. administration of CBSA-NP-hTRAIL, expression of hTRAIL mRNA was detected throughout the cerebral cortex, periventricular region, and tumor tissue (Fig. 4A ). Moreover, hTRAIL mRNA levels in brain tumor tissue were higher than in normal brain tissues. However, 24 hours after NP-hTRAIL administration, hTRAIL mRNA was mainly found in ependymal cells that line the third ventricle (periventricular region). Expression of mRNA in tumor was lower compared with those after CBSA-NP-hTRAIL administration. No hTRAIL mRNA was detected in saline control animals. At 48 hours after i.v. administration of CBSA-NP-hTRAIL, immunohistochemical analysis for the hTRAIL protein, using an antibody that did not cross-react with mTRAIL protein (31), detected specific hTRAIL protein expression in a region of the cerebral cortex inhabited by neurons and glia cells, as identified by morphology (Fig. 4B, including inset). Focal areas of hTRAIL protein expression were observed in brain tumor tissues. At 48 hours after i.v. administration of NP-hTRAIL, hTRAIL protein expression was undetectable in the cerebral cortex and periventricular region, whereas low levels of hTRAIL protein were found in brain tumor tissues. The expression hTRAIL protein in tumor tissues is detectable 12 hours after i.v. injection of CBSA-NP-hTRAIL according to immunoblotting record (Fig. 4C1). The highest level of protein expression was found 2 days after dosing. The expression-time profile can remain 6 days, suggesting sustained expression effect by using nanoparticulate vectors. However, tumor hTRAIL expression following administration of NP-hTRAIL was much weaker (Fig. 4C2). The absorbance level of hTRAIL protein 2 days after CBSA-NP-hTRAIL injection is measured 17-fold higher than that after NP-hTRAIL dosing.


Figure 4
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  		Figure 4. In vivo expression of the hTRAIL gene in normal brain and i.c. implanted C6 brain tumor. C6 cells were i.c. implanted into the right striatum of BALB/c mice. At day 7 after implantation, mice received CBSA-NP-hTRAIL, NP-hTRAIL, or saline (control) i.v. A, at 24 hours after the i.v. administration, half of the mice were anesthesized and perfused with 4% paraformaldehyde. Coronal cryostat sections were prepared of normal brain and brain tumor tissue. Using a biotin-labeled hTRAIL-specific oligonucleotide probe and the chromogen True Blue as developer, expression of hTRAIL mRNA was detected in cerebral cortex, periventricular region, and brain tumor tissue after i.v. administration of CBSA-NP-hTRAIL. Expression of hTRAIL mRNA after i.v. administration of NP-hTRAIL was lower and was observed in the periventricular region and brain tumor tissue only. B, at 48 hours after the i.v. administration, the remaining mice were sacrificed, and coronal cryostat sections were prepared of normal brain and brain tumor tissue. Expression of the hTRAIL protein was detected by binding of goat anti-human TRAIL-specific IgG followed by anti-goat IgG-Cy3 antibody (red). Cell nuclei were counterstained with DAPI. Following i.v. administration of CBSA-NP-hTRAIL, the hTRAIL protein was detected in normal brain and in focal areas in brain tumor. After i.v. injection of NP-hTRAIL, low levels of hTRAIL were found in brain tumor only. Bar, 50 µm (A and B). C, Western blotting analysis. Expression of hTRAIL protein in tumor at 0 (control), 0.5, 1, 2, 4, and 6 days following i.v. injection of CBSA-NP-hTRAIL (C1) and NP-hTRAIL (C2). Immunoblotting of ß-actin was used as internal control and confirmation of equal loading of the samples.

 
hTRAIL-mediated apoptosis was assessed by immunohistochemical analysis for active caspase-3 using an antibody that detected the cleaved p17 fragment only. At 14 days after i.v. administration of CBSA-NP-hTRAIL, p17-positive tumor cells were detected (Fig. 5A ). The TUNEL assay further detected DNA fragmentation, a marker of late apoptosis, in nuclei of tumor cells. Virtually no apoptotic cells were detected 14 days after i.v. NP-hTRAIL treatment. The antitumor effect of CBSA-NP-hTRAIL was also reflected in the median survival time of mice bearing i.c. brain tumor xenografts. After 1-week treatment, the median survival time of CBSA-NP-hTRAIL-treated mice (31.3 days) was significantly longer than that of mice treated with NP-hTRAIL (21.5 days) or saline (20.5 days; Fig. 5B; log-rank, P < 0.01). The extended administration of CBSA-NP-hTRAIL for 2 weeks can prolong the median survival time to 41.5 days (P < 0.01 compared with CBSA-NP-hTRAIL for 1-week treatment).


Figure 5
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  		Figure 5. Effects of CBSA-NP-hTRAIL and NP-hTRAIL on apoptosis and growth of i.c. implanted C6 brain tumors. C6 cells were i.c. implanted into the right striatum of BALB/c mice. The mice received 1-week treatment of CBSA-NP-hTRAIL, NP-hTRAIL, or saline (control; i.e., dosing at days 8, 10, and 12 after implantation following i.v. injection). At day 14 after implantation, mice were sacrificed for immunohistochemistry, except for 10 mice of each treatment group, which were monitored for survival. A, coronal paraffin sections of brain tumors were immunostained for the presence of active caspase-3 or DNA fragments (TUNEL). Slides were developed with diaminobenzidine and counterstained with methyl green. CBSA-NP-hTRAIL but not NP-hTRAIL induced apoptosis of brain tumor cells in vivo. Bar, 50 µm. B, Kaplan-Meier survival curve of mice harboring i.c. C6 gliomas. Besides the above 1-week treatment groups, an extended treatment group with the same dose of CBSA-NP-hTRAIL at days 8, 10, 12, 15, 17, and 19 after implantation (2-week treatment) was conducted for survival monitoring. Mice received 1-week treatment of CBSA-NP-hTRAIL, designated as CBSA-NP-hTRAIL (1w), survived significantly longer than mice that received i.v. administrations of NP-hTRAIL or saline (P < 0.01, log-rank analysis). Mice treated with CBSA-NP-hTRAIL for 2 weeks, designated as CBSA-NP-hTRAIL (2w), survived significantly longer than CBSA-NP-hTRAIL (1w) treated group (P < 0.01, log-rank analysis).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study shows for the first time tumor-specific nanoparticle delivery in the brain of an exogenous gene using a noninvasive, i.v. administration route. Fluorescence techniques developed in our laboratory showed that after i.v. administration, the nonviral vector CBSA-NP-hTRAIL crossed the BBB and accumulated in i.c. glioma xenografts via AMT. This study further showed that the encapsulated pDNA containing the hTRAIL gene was released and rapidly translocated to the nucleus, where hTRAIL was expressed and induced apoptosis of tumor cells but not normal cells.

Unlike poly(ethyleneimine) with strong proton buffering capacity, CBSA has weaker. This indicated endolysosomal escape of CBSA-NP cannot be explained as the "proton sponge effect," which caused swelling and rupture of lysosomes (32). Acid-base titration curve of CBSA indicated initial protonation of the primary amine groups followed by cationization of the carboxyl groups when increasing HCl volume. Due to the decreasing pH from physiologic to lysosomal environment, further cationization of the carboxyl groups by the excess protons from the environment being transferred to CBSA resulted in increase in positive charges of CBSA, even conversion of zeta potential of CBSA-NP-hTRAIL from negative to positive. The positive-charged CBSA-NP-hTRAIL could enhance its interaction with the negatively charged endolysosomal membrane, which probably destabilized the membrane at the point of contact, followed by extrusion into cytoplasm (33). This hypothesis was indirectly proven by the demonstration that at 30 minutes after i.v. administration, NP-hTRAIL, which has a negative zeta potential at both physiologic and acidic pH, was mostly detected in secondary endosomes and lysosomes, not in cytoplasm.

Several lines of evidence support the notion that CBSA-NP-hTRAIL passes through the BBB and tumor vascular endothelial cells by AMT. First, the electrostatic binding between CBSA-NP and the anionic micro-domains of brain capillaries could be proven by the positive immuno-gold staining results in vitro (Fig. 1A3), as heparan sulfates are the major components of negatively charged glycoproteins located in those microvessels (19, 27). Second, our findings of CSBA-NP-hTRAIL uptake by C6 glioma cells showed that this uptake was energy dependent and was specifically inhibited by free polycations, such as CBSA and polylysine. Third, we showed AMT of CBSA-NP-hTRAIL by the BBB in normal brain tissue. By using far-red fluorescent WGA, which specifically binds to glycoprotein microdomains in the brain capillary wall (27), CBSA-NP-hTRAIL and glycoproteins were found to colocalize on the luminal and abluminal membranes of the brain endothelium. CBSA-NP-hTRAIL was also detected in the endothelial cytoplasm. Fourth, we observed AMT of CBSA-NP-hTRAIL by endothelial cells of the brain tumor vasculature. Glycoproteins were found to be highly expressed in brain tumor endothelial cells and to colocalize with CBSA-NP-hTRAIL. Furthermore, TEM showed CBSA-NP to be interacting with the luminal surface of the membrane of a brain tumor endothelial cell. This is in line with a previous report that the preferential uptake of cationic molecules by angiogenic tumor epithelium occurred mostly through vesicular organelles (34). Finally, we showed that AMT of CBSA-NP-hTRAIL resulted in its uptake by brain tumor cells. In brain tumors, green fluorescent CBSA-NP-hTRAIL was detected inside red fluorescent tumor cells, but not in the interstitium. TEM showed CBSA-NP in an endocytic vesicle and free in the cytoplasm of tumor cells. CBSA-NP-hTRAIL further colocalized with glycoproteins, highly expressed by brain tumor cells. Increased tumor uptake of CBSA-NP-hTRAIL was evidenced by analysis of biodistribution (see Supplementary Data).

TRAIL selectively induces apoptosis of glioma cells via its cognate DR4 and DR5 receptors, whereas normal cells are protected by virtue of expression of the DcR1 and DcR2 antagonistic receptors (reviewed in ref. 5). In this study, TRAIL delivered via systemic administration of CBSA-NP-hTRAIL had a moderate effect on median survival of tumor-bearing mice (41 versus 22 days for control animals) compared with the increase in median survival reported for TRAIL delivered via retroviral vector (>100 versus 36 days for control animals; ref. 6). Still, retroviral vectors require i.t. delivery via craniotomy or intracarotid arterial infusion, and the associated safety risks preclude retroviral clinical use of vectors (35). The nontoxic nature of nanoparticles, on the other hand, allows repeated i.v. administrations of CBSA-NP-hTRAIL and, thus, along with the known bystander effect of TRAIL (36), improves antitumor action.

In conclusion, this study showed the feasibility of systemic administration of CBSA-NP-hTRAIL as a nonviral vector for gene therapy of glioma. The current findings encourage further studies into the application of nonviral vectors for noninvasive gene therapy of malignant glioma.


   Acknowledgments
 
Grant support: National Natural Science Foundation of China grant 30472095 (X. Jiang).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Olivia Cholewa (Molecular Probes Detection Technologies in Eugene, OR) for valuable technical assistance with fluorescent tracking and Dr. Anita Frijhoff for writing and editing contributions.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 6/28/06. Revised 9/26/06. Accepted 10/ 5/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rich JN, Bigner DD. Development of novel targeted therapies in the treatment of malignant glioma. Nat Rev Drug Discov 2004;3:430–46.[CrossRef][Medline]
  2. Rieger J, Naumann U, Glaser T, et al. APO2 ligand: a novel lethal weapon against malignant glioma? FEBS Lett 1998;427:124–8.[CrossRef][Medline]
  3. Keane MM, Ettenberg SA, Nau MM, et al. Chemotherapy augments TRAIL-induced apoptosis in breast cell lines. Cancer Res 1999;59:734–41.[Abstract/Free Full Text]
  4. Pan G, Ni J, Wei YF, et al. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 1997;277:815–8.[Abstract/Free Full Text]
  5. Hawkins CJ. TRAIL and malignant glioma. Vitam Horm 2004;67:427–52.[Medline]
  6. Roth W, Isenmann S, Naumann U, et al. Locoregional Apo2L/TRAIL eradicates intracranial human malignant glioma xenografts in athymic mice in the absence of neurotoxicity. Biochem Biophys Res Commun 1999;265:479–83.[CrossRef][Medline]
  7. Pardridge WM. The blood-brain barrier: bottleneck in brain drug development. NeuroRx 2005;2:3–14.[Abstract/Free Full Text]
  8. Culver KW, Ram Z, Wallbridge S, et al. In vivo gene transfer with retroviral vector-producer cells for treatment of experimental brain tumors. Science 1992;256:1550–2.[Abstract/Free Full Text]
  9. Nilaver G, Muldoon LL, Kroll RA, et al. Delivery of herpesvirus and adenovirus to nude rat intracerebral tumors after osmotic blood-brain barrier disruption. Proc Natl Acad Sci U S A 1995;92:9829–33.[Abstract/Free Full Text]
  10. Dewey RA, Morrissey G, Cowsill CM, et al. Chronic brain inflammation and persistent herpes simplex virus 1 thymidine kinase expression in survivors of syngeneic glioma treated by adenovirus-mediated gene therapy: implications for clinical trials. Nat Med 1999;5:1256–63.[CrossRef][Medline]
  11. McMenamin MM, Byrnes AP, Charlton HM, et al. A gamma34.5 mutant of herpes simplex 1 causes severe inflammation in the brain. Neuroscience 1998;83:1225–37.[CrossRef][Medline]
  12. Shi N, Pardridge WM. Noninvasive gene targeting to the brain. Proc Natl Acad Sci U S A 2000;97:7567–72.[Abstract/Free Full Text]
  13. Zhang Y, Jeong Lee H, Boado RJ, et al. Receptor-mediated delivery of an antisense gene to human brain cancer cells. J Gene Med 2002;4:183–94.[CrossRef][Medline]
  14. Zhang Y, Schlachetzki F, Pardridge WM. Global non-viral gene transfer to the primate brain following intravenous administration. Mol Ther 2003;7:11–8.[CrossRef][Medline]
  15. Shi N, Boado RJ, Pardridge WM. Receptor-mediated gene targeting to tissues in vivo following intravenous administration of pegylated immunoliposomes. Pharm Res 2001;18:1091–5.[CrossRef][Medline]
  16. Gref R, Minamitake Y, Perracchia MT, et al. Biodegradable long-circulating polymeric nanoparticles. Science 1994;263:1600–3.[Abstract/Free Full Text]
  17. Perez C, Sanchez A, Putnam D, et al. Poly(lactic acid)-poly(ethylene glycol) nanoparticles as new carriers for the delivery of plasmid DNA. J Control Release 2001;75:211–4.[CrossRef][Medline]
  18. Lu W, Tan YZ, Hu KL, et al. Cationic albumin conjugated pegylated nanoparticle with its transcytosis ability and little toxicity against blood-brain barrier. Int J Pharm 2005;295:247–60.[CrossRef][Medline]
  19. Lu W, Zhang Y, Tan YZ, et al. Cationic albumin-conjugated pegylated nanoparticles as novel drug carrier for brain delivery. J Control Release 2005;107:428–48.[CrossRef][Medline]
  20. Kumagai AK, Eisenberg JB, Pardridge WM. Absorptive-mediated endocytosis of cationized albumin and a ß-endorphin-cationized albumin chimeric peptide by isolated brain capillaries. Model system of blood-brain barrier transport. J Biol Chem 1987;262:15214–9.[Abstract/Free Full Text]
  21. Thole M, Nobmanna S, Huwyler J, et al. Uptake of cationzied albumin coupled liposomes by cultured porcine brain microvessel endothelial cells and intact brain capillaries. J Drug Target 2002;10:337–44.[CrossRef][Medline]
  22. Davda J, Labhasetwar V. Characterization of nanoparticle uptake by endothelial cells. Int J Pharm 2002;233:51–9.[CrossRef][Medline]
  23. Roy I, Ohulchanskyy TY, Bharali DJ, et al. Optical tracking of organically modified silica nanoparticles as DNA carriers: a nonviral, nanomedicine approach for gene delivery. Proc Natl Acad Sci U S A 2005;102:279–84.[Abstract/Free Full Text]
  24. Benns JM, Choi JS, Mahato RI, et al. pH-sensitive cationic polymer gene delivery vehicle: N-Ac-poly(L-histidine)-graft-poly(L-lysine) comb shaped polymer. Bioconjug Chem 2000;11:637–45.[CrossRef][Medline]
  25. Tang MX, Szoka FC. The influence of polymer structure on the interactions of cationic polymers with DNA and morphology of the resulting complexes. Gene Ther 1997;4:823–32.[CrossRef][Medline]
  26. Cohen H, Levy RJ, Gao J, et al. Sustained delivery and expression of DNA encapsulated in polymeric nanoparticles. Gene Ther 2000;7:1896–905.[CrossRef][Medline]
  27. Vorbrodt AW. Ultracytochemical characterization of anionic sites in the wall of brain capillaries. J Neurocytol 1989;18:359–68.[CrossRef][Medline]
  28. Panyam J, Sahoo SK, Prabha S, et al. Fluorescence and electron microscopy probes for cellular and tissue uptake of poly(D,L-lactide-co-glycolide) nanoparticles. Int J Pharm 2003;262:1–11.[CrossRef][Medline]
  29. Grataroli R, Vindrieux D, Selva J, et al. Characterization of tumour necrosis factor-{alpha}-related apoptosis-inducing ligand and its receptors in the adult human testis. Mol Hum Reprod 2004;10:123–8.[Abstract/Free Full Text]
  30. Lee M, Rentz J, Han SO, et al. Water-soluble lipopolymer as an efficient carrier for gene delivery to myocardium. Gene Ther 2003;10:585–93.[CrossRef][Medline]
  31. Wiley SR, Schooley K, Smolak PJ, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 1995;3:673–82.[CrossRef][Medline]
  32. Boussif O, Lezoualc'h F, Zanta MA, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A 1995;92:7297–301.[Abstract/Free Full Text]
  33. Panyam J, Zhou WZ, Prabha S, et al. Rapid endo-lysosomal escape of poly(DL-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery. FASEB J 2002;16:1217–26.[Abstract/Free Full Text]
  34. Thurston G, McLean JW, Rizen M, et al. Cationic liposomes target angiogenic endothelial cells in tumors and chronic inflammation in mice. J Clin Invest 1998;101:1401–13.[Medline]
  35. El-Aneed A. An overview of current delivery systems in cancer gene therapy. J Control Release 2004;94:1–14.[CrossRef][Medline]
  36. Kagawa S, He C, Gu J, et al. Antitumor activity and bystander effects of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene. Cancer Res 2001;61:3330–8.[Abstract/Free Full Text]



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