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Mol Cancer Ther. 2006;5:3162-3169
© 2006 American Association for Cancer Research

Research Articles: Therapeutics, Targets, and Development

Interleukin-13 receptor–targeted nanovesicles are a potential therapy for glioblastoma multiforme

A.B. Madhankumar1, Becky Slagle-Webb1, Akiva Mintz2, Jonas M. Sheehan1 and James R. Connor1

1 Department of Neurosurgery, Milton S. Hershey Medical Center, Penn State University, Hershey, Pennsylvania; and 2 Department of Nuclear Medicine, University of Pennsylvania, Philadelphia, Pennsylvania

Requests for reprints: James R. Connor, Department of Neurosurgery (H110), G.M. Leader Family Laboratory for Alzheimer's Disease Research, Milton S. Hershey Medical Center, Penn State University, 500 University Drive, Hershey, PA 17033-0850. Phone: 717-531-4541; Fax: 717-531-0091. E-mail: jconnor{at}psu.edu

Abstract

The difficulties associated with treatment of malignant brain tumors are well documented. For example, local infiltration of high-grade astrocytomas prevents the complete resection of all malignant cells. It is, therefore, critical to develop delivery systems for chemotherapeutic agents that ablate individual cancer cells without causing diffuse damage to surrounding brain tissue. Here, we describe sterically stable human interleukin-13 (IL-13)–conjugated liposomes, which efficiently bind to the brain cancer cells that overexpress the IL-13 receptor {alpha}2 protein. The conjugated liposomes bind to glioblastoma multiforme tissue specimens but not to normal cortex. Conjugating the liposomes with human IL-13 allows for specific binding to glioma cells and uptake of the liposomes via endocytosis. Delivering doxorubicin to glioma cells by IL-13–conjugated liposomes results in enhanced cytotoxicity and increased accumulation and retention of drug in the glioma cells compared with delivery of free drug. The therapeutic potential and targeting efficacy of the IL-13–conjugated liposomes carrying doxorubicin was tested in vivo using a s.c. glioma tumor mouse model. Animals receiving i.p. injections of IL-13–conjugated liposomes carrying doxorubicin for 7 weeks had a mean tumor volume of 37 mm3 compared with a mean volume of 192 mm3 in animals injected with nontargeted liposomes. These results strongly suggest that IL-13–conjugated liposomes carrying cytotoxic agents are a feasible approach for creating a nanovesicle drug delivery system for brain tumor therapy. [Mol Cancer Ther 2006;5(12):3162–9]

Introduction

Human interleukin-13 (IL-13) is a cytokine secreted by activated T cells that elicits both proinflammatory and anti-inflammatory immune responses (1, 2). IL-13 has two types of receptors: IL-13/4R, which is present in normal cells and whose binding is shared with IL-4, and IL-13R{alpha}2, which does not bind IL-4 (3). IL-13R{alpha}2 is associated with high-grade astrocytomas more commonly referred to as glioblastoma multiforme (GBM) and is not significantly expressed in normal tissue, with the exception of the testes (3–5). A recent study determined that pilocytic astrocytomas, the most common astrocytic tumors in children, also overexpress the IL-13R{alpha}2 receptor (6). Thus, the IL-13R{alpha}2 receptor is an excellent potential target for delivering cytotoxic agents to a variety of devastating brain tumors.

A number of attempts to use IL-13R{alpha}2 as a target for high-grade astrocytoma therapy have been reported both in vitro and in vivo. Some of the successful modalities used include IL-13–based cytotoxins (7, 8), IL-13R{alpha}2–targeted viruses (9), and IL-13R{alpha}2 immunotherapy (10, 11). One significant way to enhance the therapeutic index of the anticancer drugs is to specifically deliver these agents directly to the tumor cells through a carrier, thereby keeping them away from healthy cells that are sensitive to toxic effects of the drugs. Such target-oriented delivery systems include colloidal delivery systems such as microspheres, nanoparticles, liposomes, and micelles. Liposomes are considered a promising drug-delivery technology for brain tumor therapy (12–16).

Most of the existing liposome-based anticancer therapies use nontargeted delivery and display a series of toxic side effects to normal cells (17). However, it is thought that targeted delivery of liposomes should result in increased accumulation and retention of liposomes at the tumor site, thus decreasing the systemic toxicity and increasing the therapeutic ability of liposome-based therapy. Conjugation of functional proteins or monoclonal antibodies to liposomes has been extensively explored, primarily to target specific cells or tissues (16, 18–20). Our experience in formulating colloidal particulate carriers like micelles and microspheres (21–23) for drug delivery applications motivated us to design IL-13–conjugated liposomes for site-specific delivery of drugs and diagnostic agents for brain tumor therapy. Apart from drug targeting, drug transport to the solid tumors is another area gaining significance due to several factors such as multidrug resistance and P-glycoprotein–mediated drug efflux.

In the present work, we developed IL-13–conjugated liposomes to deliver chemotherapeutic agents specifically to brain tumors. For our studies, we use doxorubicin as an antineoplastic agent that is widely used for intracranial glioma models in animals (24–27). To establish proof of our concept, we examined the binding ability of the IL-13–conjugated liposomes in a cell culture model and their cytotoxic potential in a glioma cell line. Their ability to overcome P-glycoprotein–mediated expulsion of drug was also studied. The cell culture experiments yielded positive results. Therefore, the antitumor effect of doxorubicin-encapsulated, IL-13–conjugated liposomes was evaluated on a s.c. tumor mouse model.

Materials and Methods

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene-glycol)2000] (ammonium salt), dipalmitoylphosphatidylcholine, cholesterol, and L-{alpha}-phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) were purchased from Avanti Polar Lipids (Alabaster, AL). Stearylamine was purchased from Sigma Chemical (St. Louis, MO). Human U251 and U87 glioma cells were purchased from American Type Tissue Culture Collection (Manassas, VA). Doxorubicin was obtained from Sigma Chemical. Corning 96-well plates were purchased from Corning (Corning, NY); cyclosporine A was from Calbiochem (La Jolla, CA); and early endosomal antigen 1 (EEA1) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA).

Preparation and Characterization of IL-13–Conjugated Liposomes
Sterically stable liposomes were formulated by using dipalmitoylphosphatidylcholine, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene-glycol)2000], and N-[3-(2-pyridyldithio)-propinyl]stearylamine (molar ratio of 10:5:1.5:1.5) dissolved in methanol/chloroform mixture (2:1; ref. 28). The liposomes were subsequently roto-evaporated to obtain a lipid film, which was further dried in a desiccator. For doxorubicin encapsulation or binding studies using HEPES buffer, the lipid film was hydrated in 155 mmol/L ammonium sulfate (pH 5.5) and then sonicated in a bath-type sonicator for 15 min. To fluorescently tag the liposomes for the cellular uptake, the liposomes were constructed using 1 mol% of fluorescently labeled phospholipid [L-{alpha}-phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt)]. A polycarbonate membrane of gradually decreasing pore size was used to produce small unilamellar vesicles by extruding through two-stacked, 0.1-µm polycarbonate membrane and subsequently with 0.05-µm polycarbonate membrane using a nitrogen pressure–operated extruder (Lipex extruder, Northern Lipids, Inc., Vancouver, British Columbia, Canada). All the extrusions were done at an operating pressure of 800 p.s.i. (5,440 kPa). The liposomes were then purified and sterilized by passing through Sephadex G25M column. The liposome concentration was determined by phosphate assay (29). The size distribution of the liposomes was determined by dynamic light scattering, which was conducted using an ALV/DLS/SLS-5022F compact goniometer system (ALV, Langen, Germany), which was confirmed by transmission electron microscopy using uranyl acetate as the staining agent.

Human IL-13 gene, which was isolated from the total RNA (BD Biosciences, Mountain View, CA) from human testis by reverse transcription-PCR, which was cloned into the TOPO vector (Invitrogen, Carlsbad, CA), expressed in Escherichia coli as His-tagged protein and purified by nickel affinity binding. Conjugation of IL-13 to liposomes was done according to the method reported by Singh et al. (28). A heterobifunctional reagent, N-succinimidyl-3(2-pyridyldithio)propionate, was used to introduce pyridyl disulfide groups to the IL-13 molecule (28). Briefly, 10 mol of N-succinimidyl-3(2-pyridyldithio)propionate was dissolved in methanol and then this solution was reacted with 1 mol of IL-13 in PBS for 24 h at 4°C. The unreacted N-succinimidyl-3(2-pyridyldithio)propionate was removed by dialysis against PBS (molecular weight cutoff, 10,000). The dialysate was reduced with DTT (20 mmol/L final concentration), and the unreacted excess DTT was removed by gel filtration through a Sephadex G25M column. Thiolation of IL-13 was verified by the presence of free sulfhydryl groups, which were estimated by Ellman's method (30) according to Ellman's reagent protocol (Pierce, Rockford, IL).

Thiolated IL-13 protein was slowly added to a 5-mL beaker containing the liposomes and a magnetic stirring bar and incubated overnight with slow stirring at 4°C. The conjugated liposomes were separated by ultracentrifugation at 40,000 rpm. The IL-13 to phospholipid mole ratio was maintained at 1:700. After conjugation, the presence of IL-13 protein on the liposomes were verified by Coomassie (Bradford; Pierce) protein binding assay. The lipid content of the liposome was measured by phosphorus estimation according to the method of Morrison (31).

To add transferrin to the liposomes, commercially available bovine Tf (Sigma Chemical) was conjugated to IL-13 liposomes by a similar method to that described for IL-13. An N-succinimidyl-3(2-pyridyldithio)propionate–modified Tf protein was reduced with DTT for thiolation. Thiolated Tf was reacted with IL-13–conjugated liposomes using a phospholipid to Tf mole ratio of 1:700 using the same methods and conditions as that for IL-13 conjugation.

Method of Encapsulation of Doxorubicin in the Liposomes
Doxorubicin was encapsulated into the liposomes by ammonium sulfate gradient method (32). The liposomes were hydrated with ammonium sulfate (pH 5.5; 155 mmol/L) using a bath-type sonicator. The liposomes were then extruded as before. The concentration of phospholipid was maintained at 10 mmol/L. The external buffer was exchanged by passing the liposomes through Sephadex G-25M column and eluting them with 123 mmol/L sodium citrate (pH 5.5). Then, the liposomes were incubated with doxorubicin (0.2 mg doxorubicin per milligram of phospholipid) for 1 h at 65°C. In all our preparations, the drug to lipid weight ratio was maintained as 1:5. Unencapsulated doxorubicin was removed by passing the liposomes through Sephadex G25M column and exchanging them with PBS.

Doxorubicin Leakage Study
The leakage of doxorubicin from the IL-13–conjugated liposomes and the unconjugated liposomes was determined by suspending 10 µL of doxorubicin-containing liposomes in 0.5 mL of human serum or dialyzed against a large volume of PBS. Both experiments were done at 37°C for increasing time intervals. Both serum and PBS media were evaluated to compare shelf life (PBS) and in vivo stability for delivery (serum). To determine the amounts of doxorubicin that may have been released from the liposomes, the serum samples were centrifuged at 40,000 rpm and the supernatants were analyzed for doxorubicin by measuring the absorbance at 492 nm. For the PBS studies, the dialysate was collected and assayed for doxorubicin.

Uptake of IL-13–Conjugated Liposomes in Normal and Glioma Cells
Uptake of the IL-13–conjugated liposomes on glioma cells was done to investigate the ability of the glioma cells to internalize the liposomes. Both U251 and U87 glioma cells (10,000 cells each) were cultivated on a chamber slide for 24 h. IL-13–conjugated, rhodamine-labeled liposomes were added for 120 min at 37°C. Human umbilical vein endothelial cells and SVGp12 glial cells (purchased from American Type Culture Collection) served as controls. The SVG p12 cell lines are human fetal glial cells from brain material, which are transfected with DNA from an ori-mutant of SV40. The cells were washed thrice with PBS to end the exposure to liposomes and then viewed with a confocal microscope. The cells were stained with 4',6-diamidino-2-phenylindole to visualize the nuclei.

To determine if the uptake of the liposomes is involved the endosomal system, U251 glioma cells were cultured on chamber slides as described above and the cells were permeabilized and blocked for 30 min in 0.1% bovine serum albumin and PBS (blocking buffer). The cells were treated with rhodamine-labeled IL-13–conjugated liposomes and then stained with polyclonal EEA1 antibody (1:15) for 30 min. The cells were then washed thrice with PBS and counterstained with FITC–anti-goat antibody (1:75) for 30 min and observed under fluorescent microscope. The images were captured using a digital camera.

Flow Cytometry
Flow cytometry was used to measure total intracellular doxorubicin fluorescence (33). In this report, we refer to fluorescence intensity as intracellular drug content. Cells (1 x 106) were exposed to 20 µmol/L of drug as (a) free doxorubicin; (b) doxorubicin-encapsulated, IL-13–conjugated liposomes; and (c) doxorubicin-encapsulated unconjugated liposomes for 2 h. All drug treatments and posttreatment incubations were done in complete growth medium. The cells were washed to remove any free adherent doxorubicin using PBS and centrifuged. Cells were released from tissue culture dishes with 0.05% trypsin/0.02% EDTA followed by PBS washing (centrifugation, 5 min, 500 x g) and resuspended in PBS for flow cytometry assay. The intracellular accumulation of inherently fluorescent doxorubicin was evaluated using a fluorescence activated cell analyzer. A single 15-mW argon ion laser beam (488 nm) was used to excite the fluorescence of doxorubicin. A total of 10,000 cells were analyzed for each histogram. Experiments were repeated thrice and the fluorescence intensities of doxorubicin were expressed in arbitrary units.

Binding to Human Brain Tumor Sections
To show the potential clinical application of the conjugated liposomes, we obtained GBM and pilocytic astrocytoma brain tumor sections and exposed them to the rhodamine-labeled IL-13 liposomes. Brain tumor samples were obtained from patients undergoing surgical decompression at Penn State University Hershey Medical Center. All studies involving human specimens were approved by the respective Human Subjects Protection Office at the Penn State College of Medicine (protocol no. 96-123EP). Serial tissue sections were generated (10 µm) on a cryostat, thaw mounted on chromalum-coated slides, and stored at –70°C until analyzed. The sections were then blocked with normal goat serum (10%) and then exposed to rhodamine-labeled, IL-13–conjugated liposomes for 1 h at 37°C. Then, sections were washed thrice with PBS before observing them via fluorescence microscopy. To test the hypothesis that the IL-13–conjugated liposomes interacted with the IL-13 receptor on the GBM tumors, some of the GBM sections were blocked with 1 mg/mL concentration of IL-13R{alpha}2 receptor antibody and followed by exposure to rhodamine-labeled, IL-13–conjugated liposomes. The sections were then washed with PBS and observed under a fluorescence microscope.

Effect of P-Glycoprotein Inhibitor on the Internalization of IL-13–Conjugated Liposomes in the Glioma Cells
About 50,000 U251 glioma cells were plated in a small Petri dish and were exposed to either IL-13–conjugated liposomes carrying 20 µmol/L of doxorubicin or to the same concentration of free doxorubicin for 2 h. The cells in each condition were either treated or not with cyclosporine A, a P-glycoprotein inhibitor (5 µg/mL), for 30 min before addition of the liposomes. After 2-h incubation, cells were washed with PBS, removed with versene, and subjected to flow cytometry.

Cytotoxicity Assay with Doxorubicin-Encapsulated, Ligand-Targeted Liposomes
In our experiments, we used doxorubicin-encapsulated liposomes, which are unconjugated, conjugated with IL-13, or double conjugated with IL-13 and Tf, to determine their cytotoxic potential. Cytotoxicity was measured after adding serially diluted doxorubicin-encapsulated liposomes to U251 glioma cells plated in 96-well cell culture plates at a concentration of 5 x 103 per well. Cell survival was determined after 48 h by 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium/phenazine methosulfate assay. Cells treated with high concentrations of cycloheximide served as the background for the assay (34, 35).

In vivo Therapeutic Efficacy of Targeted Liposomes
To test the in vivo efficacy of the targeted liposomal system, adult female athymic nude mice were implanted in the flank s.c. with U251 glioma cells. Exponentially growing cells were harvested and 15 x 106 cells per mouse were s.c. injected. After 2 weeks, a tumor of volume 14 to 30 mm3 was observed. At that time, the mice were divided into five different groups of six mice in each group. One group of mice was injected with IL-13–conjugated liposomes carrying doxorubicin at a dosage of 15 mg/kg of body weight. A second group was injected with the same amount of liposomes carrying 15 mg/kg body weight of doxorubicin, but these liposomes were unconjugated. A third group received injections of IL-13–conjugated liposomes but with a lower dosage of doxorubicin (7.5 mg/kg of body weight). The fourth group of mice were untreated and received injections of 0.1 mol/L PBS as a control. A fifth group of mice were injected with unconjugated liposomes carrying no drug as an additional control. All the drugs were administered i.p. weekly. The injections were given opposite the side of the s.c. tumor. The tumor size, health, and survival of the mice were monitored weekly by an investigator (B.W.) blinded as to the groups of mice. These experiments were approved by the Pennsylvania State University Institutional Animal Care and Use Committees.

Results

Liposome Composition and Particle Size
The particle size of the liposome as confirmed by laser particle size analyzer and transmission electron microscopy was found to be in the range of 50 to 150 nm with a mean size of 104 nm. The polydispersity index for various batches of nanovesicles consistently lies in the range of 0.2 to 0.4. After conjugation and purification, the concentration of the phospholipids in the liposome was 21.8 µg of phospholipids per microliter and the concentration of IL-13 conjugated on the liposomes after doxorubicin encapsulation was 3.46 x 10–7 µmol of IL-13 per microgram of phospholipids. The final concentration of doxorubicin is 0.18 µg/µg of phospholipid.

We also observed the effect of temperature on encapsulation efficiency of doxorubicin to be maximum (90%) at 65°C when compared with lower temperatures, 25°C and 40°C, where the encapsulation efficiencies are 45% and 72%, respectively. The T1/2 for doxorubicin leakage from IL-13 liposome at 37°C in PBS was 25 days, whereas with unconjugated liposomes, the T1/2 is ~45 days. Thus, the IL-13–conjugated liposomes were not substantially leaky during the experimental period, because our experiments were done within 2 weeks of preparation. We did not observe any significant leakage of doxorubicin from the liposomes that were incubated in human serum at 37°C for at least 1 week.

Binding to Glioma Cells
For IL-13 receptor–targeted liposomes to be considered for clinical use, it is necessary to show that glioma cells take up the liposomes. Figure 1 shows the uptake of the liposomes in both U87 and U251 glioma cell lines, whereas normal cells like human umbilical vein endothelial cells and the immortalized glial cell line SVGp12, which do not overexpress IL-13 receptor, had no detectable uptake over the same exposure time (36).


Figure 1
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  		Figure 1. Uptake of IL-13–conjugated rhodamine-labeled liposomes in human cell lines using rhodamine-labeled liposomes. Uptake is clearly visible in the U251 and U87 glioma cells. Red, rhodamine staining; blue, 4',6-diamidino-2-phenylindole nuclear staining. SVGp12 and human umbilical vein endothelial cells (HUVEC) served as negative controls. The cells were exposed to the liposomes for 2 h.

 
Intracellular Accumulation and Retention of Doxorubicin in U251 Glioma Cells
The uptake and accumulation of the IL-13–conjugated liposomes were analyzed using flow cytometry and fluorescent microscopy. The IL-13–conjugated liposomes enter early endosomes as shown by colocalization with EEA1 (Fig. 2 ). The relative accumulation of the doxorubicin in U251 cells depending on the mode of delivery was shown by flow cytometry (Fig. 3 ). The ability to show doxorubicin in cells by fluorescence-activated cell sorting (FACS) analysis takes advantage of the intrinsic fluorescence of doxorubicin when excited at 488 nm (37, 38). The flow cytometry analysis showed a right shift in the curve, indicating an increase in the cell fluorescence in U251 glioma cells after exposure to free doxorubicin or liposomal doxorubicin. The right shift is greater with IL-13–conjugated liposomal doxorubicin than with nonconjugated liposomes (Fig. 3A). It is well known that drug accumulation in the cancer cells is decreased by P-glycoprotein activity (39–41). When doxorubicin is delivered by IL-13–conjugated liposomes, the intrinsic fluorescence of the doxorubicin accumulated or retained intracellularly in U251 glioma cells is much higher than that seen in cells exposed to free doxorubicin (Fig. 3B). Indeed, the level of doxorubicin detected in the cells following delivery via liposome was even greater than that seen in cells treated with free doxorubicin, which were also exposed to cyclosporine A, a P-glycoprotein inhibitor (Fig. 3B).


Figure 2
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  		Figure 2. Colocalization of IL-13–conjugated, rhodamine-labeled liposomes with EEA1 using U251 glioma cells. The cells were exposed to the liposomes and subsequently with EEA1 for 40 min. A, IL-13 liposomal uptake (red). B, cells were exposed to EEA1 and then labeled with FITC-conjugated anti-goat IgG for 1 h (1:100; green). C, superimposed image of cells in A and B showing the colocalization of liposomes with EEA1.

 

Figure 3
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  		Figure 3. Flow cytometric analysis of doxorubicin accumulation in U251 glioma cells. A, retention of doxorubicin in U251 glioma cells after exposure to doxorubicin in unconjugated liposomes (LIPDXR) and doxorubicin in IL-13–conjugated liposomes (IL13LIPDXR). Encapsulating doxorubicin in IL-13–conjugated liposomes results in an increase accumulation of doxorubicin over 2 h of exposure. B, U251 glioma cells express P-glycoproteins and are a good model for examining multidrug resistance. Therefore, we compared the accumulation of free doxorubicin in the presence (DXR-Cyclo) or absence (DXR) of cyclosporine A, a P-glycoprotein inhibitor. Cells were exposed to doxorubicin for 2 h. The presence of cyclosporine A resulted in retention of doxorubicin to levels that were similar to the IL-13 liposome–delivered doxorubicin. Histograms are data from one representative experiment. The experiments were done thrice for each condition. Control in each condition are cells alone with no treatment.

 
Exposure of Glioma Tumors to Liposomes
Representative samples of GBM, pilocytic astrocytomas, and normal human cortex exposed to IL-13–conjugated liposomes tagged with rhodamine are shown in Fig. 4 . There was a much greater affinity of the GBM and pilocytic astrocytoma samples for the IL-13–conjugated liposomes than the medulloblastoma or normal human cortex samples. The specificity of this association of IL-13–conjugated liposomes to the IL-13 receptor was shown by exposing the tumor sections to IL-13 receptor antibody followed by IL-13–conjugated vesicles. This approach resulted in a decrease in the binding of IL-13–conjugated, rhodamine-labeled liposomes.


Figure 4
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  		Figure 4. Affinity of rhodamine-labeled IL-13–conjugated liposomes for brain tissue samples. A number of brain tissue samples (normal and tumor) were examined for affinity to the IL-13–conjugated liposomes. The results show highest affinities of the liposomes from GBM and relatively less, but still detectable, affinities from pilocytic astrocytoma. No binding is observed to medulloblastoma or to the normal human cortex. To show that the IL-13–conjugated liposomes have affinity for the IL-13 receptor, a GBM tissue section (GBM#15) was first exposed to IL-13 receptor antibody and then exposed to the liposomes. This approach resulted in a decrease in binding of the GBM with the liposomes.

 
Cytotoxicity Assay with Ligand-Targeted Liposomes
The cytotoxicity of doxorubicin on U251 glioma cells encapsulated in IL-13–conjugated liposomes versus unconjugated liposomes was compared and the results are shown in Fig. 5 . Because we were also evaluating the possibility of using liposomes doubly conjugated with Tf and IL-13, these liposomes were also included in this experiment. The concentration of liposomes, which were added to each of the cell cultures, was identical; each liposome carried equal amounts of doxorubicin. At the lowest concentration, 150 ng/mL of liposomal doxorubicin, IL-13–conjugated liposomes were 31.7% more cytotoxic than unconjugated liposomes (P < 0.001). The cytotoxicity of the doubly conjugated (IL-13, Tf) liposomes was similar to the IL-13–conjugated liposomes (35.3%). With increasing concentration, the liposomal doxorubicin cytotoxicity increases at a faster rate than the cytotoxicity associated with the unconjugated liposomes.


Figure 5
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  		Figure 5. Cytotoxicity assay of IL-13– and transferrin-conjugated liposomes carrying doxorubicin on U251cells. The cytotoxic potential of ligand-targeted liposomes is, in general, higher at each concentration of doxorubicin than the unconjugated liposomes carrying the same amount of doxorubicin. The presence of transferrin does not effect the toxicity of the liposomes. Statistical significance was determined by ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
In vivo Anticancer Therapeutic Efficacy
The tumors in the control mice grew from 14 to 570 mm3 in 7 weeks, whereas the tumor growth rate is much lower in animals that received doxorubicin-carrying liposomes. The most effective approach at reducing the tumors was IL-13–conjugated liposomes carrying doxorubicin (15 mg/kg of body weight). In this group, the tumor volume decreased by 69% over the first 2 weeks after injections (Fig. 6 ). The only other group to show an initial decrease in tumor size (52%) was the one receiving injections of unconjugated liposomes carrying doxorubicin. The group receiving the highest dose of the conjugated liposomes and doxorubicin had a tumor volume of only 37 mm3 or <10% of the untreated group after 7 weeks (termination of the experiment). Animals receiving the same dose of unconjugated liposomes had a tumor volume of 192 mm3 in 7 weeks, 5-fold more than the animals receiving the same concentration of doxorubicin in targeted liposomes and 22% higher than animals receiving the lowest dose of doxorubicin in conjugated liposomes (Fig. 6). In the group that received only liposomes (untargeted and not containing doxorubicin), the tumor volume did not decrease appreciably and at the end of 7 weeks had an average volume of 452 mm3. During the course of these studies, only one animal died. This animal was in the high doxorubicin group (conjugated liposomes) and the death seemed related to an injection artifact. No animals died in the other groups.


Figure 6
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  		Figure 6. The therapeutic efficacy of the IL-13 receptor–targeted liposomes carrying doxorubicin was tested in a s.c. glioma tumor model in nude mice. Mice were given i.p. injections weekly. A, mice receiving targeted liposomes with doxorubicin (DXR) had a greater reduction in tumor size in the first 2 wks compared with the animals receiving the same concentration of unconjugated liposomes and doxorubicin. The tumors in all of the other groups increased during the initial 3 wks of injections. B, pattern of tumor growth over 7 wks of injections of liposomes (LIP) containing doxorubicin at the indicated concentrations or liposomes without drug (LIP without DXR). Points, mean tumor volume; bars, SE. Error bars on the LIP (DXR) 15 mg/kg group are contained within the symbol for this group.

 
Discussion

Previously, IL-13 receptors have been identified as a potential target on high-grade astrocytomas, but the outcomes have been mixed. Here, we show an alternative approach of using IL-13–conjugated liposomes to selectively target and deliver the cytotoxin doxorubicin to tumor cells that is effective in both in vivo and cell culture models. The liposomes in this study have a mean size of 104 nm. This size is optimal for nanoparticles to cross the blood-brain barrier (42, 43); moreover, smaller liposomes have a relatively extended half-life (44). In addition, the antitumor activity of the liposomal doxorubicin is sensitive to vesicle size, and the liposomes in this size range can readily release their contents within the cells (45), which is consistent with our observations in this study.

Our liposome system is composed of polyethylene glycol lipids that provide a steric barrier at the liposome surface, inhibiting protein binding and therefore opsonization (46, 47). The ligand IL-13 is conjugated to the lipid portion rather than on the surface of the polyethylene glycol moiety. Our data indicate that the liposomes constructed in this manner maintain their targeting property, maintain an ability to effectively encapsulate and retain the drug following covalent attachment of IL-13, and are still able to bind the target IL-13R{alpha}2 on glioma cells. Moreover, our liposomes are relatively stable and, unlike egg-phosphatidylcholine/cholesterol liposomes, after drug encapsulation, they are not leaky in serum or buffer at physiologic temperatures. The liposomes configured in our study only became leaky at a temperature well above the transition temperature of dipalmitoylphosphatidylcholine (48).

Our study showed effective binding of IL-13–conjugated liposomes to the malignant glioma cells and the clinical specimens of brain tumors in situ. We provided evidence for an affinity to high-grade astrocytoma (GBM) as well as a low-grade pilocytic astrocytoma. These observations are consistent with a recent report where the presence of IL-13R{alpha}2 was shown on these tumors (6). The uptake studies showed that the liposomes were found in early endosomes, which is consistent with receptor-mediated uptake. The lack of affinity of the liposomes for the normal human cortex or the human umbilical vein endothelial cells is consistent with the absence of detectable IL-13R{alpha}2 receptor.

The cytotoxicity experiments and in vivo experiments revealed that the IL-13–conjugated liposomes were superior to the unconjugated liposomes at killing the tumor cells. Most brain tumors express P-glycoprotein, which confers drug resistance to glioma cells (49). Our results showed that the liposome-delivered doxorubicin was not expelled by P-glycoprotein from the cell, unlike the unencapsulated doxorubicin. Therefore, the explanation for the enhanced cytotoxicity with the IL-13–targeted liposomes is that doxorubicin delivered by these liposomes results in increased accumulation and retention in glioma cells. The demonstration that liposome-delivered doxorubicin can avoid expulsion from tumor cells in a cell culture model while having greater efficacy in the in vivo model strongly supports the notion that liposomal delivery is a viable option for brain tumors in vivo.

A critical component of drug delivery systems is their ability to target the tumors without adverse effect to the normal healthy tissues and to transport therapeutic agents into the tumors overcoming the P-glycoprotein–mediated drug resistance. In our in vivo model, we could clearly observe higher therapeutic efficacy of the IL-13–conjugated liposomes where the tumor volume was reduced by 68% in 3 weeks, whereas in the unconjugated liposomes the tumor volume was only reduced by 50% over 3 weeks. The difference in final volume (7 weeks) between conjugated and nonconjugated liposomes (over 500%) is compelling evidence that IL-13–conjugated liposomes carrying doxorubicin are much more efficacious than untargeted liposomes carrying the same amount of doxorubicin. The cell culture data suggest that the greater efficacy of the targeted liposomes is a combination of the receptor targeting nature of the liposomes and the ability of the targeted liposomes to overcome the P-glycoprotein–mediated drug efflux by the tumor. Thus, IL-13 receptor–targeted nanovesicles represent a viable approach where the liposomes of particle size ranging from 50 to 150 nm can be used to deliver chemotherapeutic agents to brain tumor cells and may be a viable option for i.v. drug delivery applications across the blood-brain barrier.

Footnotes

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.

Received 8/10/06; revised 9/30/06; accepted 10/27/06.

References

  1. McKenzie AN, Culpepper JA, de Waal Malefyt R, et al. Interleukin 13, a T-cell-derived cytokine that regulates human monocyte and B-cell function. Proc Natl Acad Sci U S A 1993;90:3735–9.[Abstract/Free Full Text]
  2. Minty A, Chalon P, Derocq JM, et al. Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses. Nature 1993;362:248–50.[CrossRef][Medline]
  3. Caput D, Laurent P, Kaghad M, et al. Cloning and characterization of a specific interleukin (IL)-13 binding protein structurally related to the IL-5 receptor {alpha} chain. J Biol Chem 1996;271:16921–6.[Abstract/Free Full Text]
  4. Debinski W, Slagle B, Gibo DM, Powers SK, Gillespie GY. Expression of a restrictive receptor for interleukin 13 is associated with glial transformation. J Neuro-oncol 2000;48:103–11.[CrossRef][Medline]
  5. Debinski W, Gibo DM. Molecular expression analysis of restrictive receptor for interleukin 13, a brain tumor-associated cancer/testis antigen. Mol Med 2000;6:440–9.[Medline]
  6. Kawakami M, Kawakami K, Takahashi S, Abe M, Puri RK. Analysis of interleukin-13 receptor {alpha}2 expression in human pediatric brain tumors. Cancer 2004;101:1036–42.[CrossRef][Medline]
  7. Nash KT, Thompson JP, Debinski W. Molecular targeting of malignant gliomas with novel multiply-mutated interleukin 13-based cytotoxins. Crit Rev Oncol Hematol 2001;39:87–98.[Medline]
  8. Husain SR, Puri RK. Interleukin-13 receptor-directed cytotoxin for malignant glioma therapy: from bench to bedside. J Neuro-oncol 2003;65:37–48.[CrossRef][Medline]
  9. Zhou G, Ye GJ, Debinski W, Roizman B. Engineered herpes simplex virus 1 is dependent on IL13R{alpha}2 receptor for cell entry and independent of glycoprotein D receptor interaction. Proc Natl Acad Sci U S A 2002;99:15124–9.[Abstract/Free Full Text]
  10. Mintz A, Gibo DM, Madhankumar AB, Debinski W. Molecular targeting with recombinant cytotoxins of interleukin-13 receptor {alpha}2-expressing glioma. J Neuro-oncol 2003;64:117–23.[CrossRef][Medline]
  11. Kawakami M, Kawakami K, Puri RK. Intratumor administration of interleukin 13 receptor-targeted cytotoxin induces apoptotic cell death in human malignant glioma tumor xenografts. Mol Cancer Ther 2002;1:999–1007.[Abstract/Free Full Text]
  12. Martuza RL. Molecular neurosurgery for glial and neuronal disorders. Stereotact Funct Neurosurg 1992;59:92–9.[Medline]
  13. Okada H, Okamoto S, Yoshida J. [Gene therapy for brain tumors: cytokine gene therapy using DNA/liposome (series 3)]. No Shinkei Geka 1994;22:999–1004.[Medline]
  14. Sharma US, Sharma A, Chau RI, Straubinger RM. Liposome-mediated therapy of intracranial brain tumors in a rat model. Pharm Res 1997;14:992–8.[CrossRef][Medline]
  15. Siegal T, Horowitz A, Gabizon A. Doxorubicin encapsulated in sterically stabilized liposomes for the treatment of a brain tumor model: biodistribution and therapeutic efficacy. J Neurosurg 1995;83:1029–37.[Medline]
  16. Kakinuma K, Tanaka R, Takahashi H, Watanabe M, Nakagawa T, Kuroki M. Targeting chemotherapy for malignant brain tumor using thermosensitive liposome and localized hyperthermia. J Neurosurg 1996;84:180–4.[Medline]
  17. Sapra P, Allen TM. Ligand-targeted liposomal anticancer drugs. Prog Lipid Res 2003;42:439–62.[CrossRef][Medline]
  18. Huwyler J, Cerletti A, Fricker G, Eberle AN, Drewe J. By-passing of P-glycoprotein using immunoliposomes. J Drug Target 2002;10:73–9.[CrossRef][Medline]
  19. Suzuki S, Uno S, Fukuda Y, Aoki Y, Masuko T, Hashimoto Y. Cytotoxicity of anti-c-erbB-2 immunoliposomes containing doxorubicin on human cancer cells. Br J Cancer 1995;72:663–8.[Medline]
  20. Uyama I, Kumai K, Yasuda T, et al. Improvement of therapeutic effect by using Fab' fragment in the treatment of carcinoembryonic antigen-positive human solid tumors with adriamycin-entrapped immunoliposomes. Jpn J Cancer Res 1994;85:434–40.[CrossRef][Medline]
  21. Madhan Kumar AB, Panduranga Rao K. Poly(palmitoyl-hydroxyproline ester) microspheres as potential oral controlled drug delivery system. Int J Pharm 1997;149:107–14.[CrossRef]
  22. Madhan Kumar AB, Panduranga Rao K. Preparation and characterization of pH-sensitive proteinoid microspheres for the oral delivery of methotrexate. Biomaterials 1998;19:725–32.[CrossRef][Medline]
  23. Madhankumar AB, Jayakumar R, Panduranga Rao K. Synthesis and aggregational behavior of acidic proteinoid. J Polym Sci [A1] 1996;34:2915–24.[CrossRef]
  24. Lesniak MS, Upadhyay U, Goodwin R, Tyler B, Brem H. Local delivery of doxorubicin for the treatment of malignant brain tumors in rats. Anticancer Res 2005;25:3825–31.[Medline]
  25. Dorr RT, Von Doff DD. Doxorubicin. In: Dorr RT, Von Doff DD, editors. Cancer chemotherapy handbook. Norwalk (CT): Appleton & Lange; 1994. p. 395–416.
  26. Muldoon LL, Neuwelt EA. BR96-DOX immunoconjugate targeting of chemotherapy in brain tumor models. J Neurooncol 2003;65:49–62.[CrossRef][Medline]
  27. Saul JM, Annapragada A, Natarajan JV, Bellamkonda RV. Controlled targeting of liposomal doxorubicin via the folate receptor in vitro. J Control Release 2003;92:49–67.[CrossRef][Medline]
  28. Singh M, Ferdous AJ, Kanikkannan N, Faulkner G. Stealth monensin immunoliposomes as potentiator of immunotoxins in vitro. Eur J Pharm Biopharm 2001;52:13–20.[CrossRef][Medline]
  29. Morrison WR. Anal Biochem 1964;7:281–4.[CrossRef]
  30. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70–7.[CrossRef][Medline]
  31. Morrison WR. A fast, simple and reliable method for the microdetermination of phosphorus in biological materials. Anal Biochem 1964;7:218–24.[CrossRef][Medline]
  32. Hansen CB, Kao GY, Moase EH, Zalipsky S, Allen TM. Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of coupling procedures. Biochim Biophys Acta 1995;1239:133–44.[Medline]
  33. Krishan A, Ganapathi R. Laser flow cytometry and cancer chemotherapy: detection of intracellular anthracyclines by flow cytometry. J Histochem Cytochem 1979;27:1655–6.[Abstract]
  34. Madhankumar AB, Mintz A, Debinski W. Alanine-scanning mutagenesis of {alpha}-helix D segment of interleukin-13 reveals new functionally important residues of the cytokine. J Biol Chem 2002;277:43194–205.[Abstract/Free Full Text]
  35. Thompson JP, Debinski W. Mutants of interleukin 13 with altered reactivity toward interleukin 13 receptors. J Biol Chem 1999;274:29944–50.[Abstract/Free Full Text]
  36. Hu N, Gibo DM, Debinski W. Cytokine up-regulation of IL13R{alpha}2 in GBM cells leads to an increased potency of recombinant IL13 cytotoxin. Cancer Ther 2005;3:531–42.
  37. Krishan A, Ganapathi R. Laser flow cytometric studies on the intracellular fluorescence of anthracyclines. Cancer Res 1980;40:3895–900.[Abstract/Free Full Text]
  38. Frankfurt OS. Flow cytometric screening for selective toxicity to multidrug-resistant cells. J Natl Cancer Inst 1987;79:831–6.[Medline]
  39. Gottesman MM, Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem 1993;62:385–427.[CrossRef][Medline]
  40. Inaba M, Kobayashi H, Sakurai Y, Johnson RK. Active efflux of daunorubicin and Adriamycin in sensitive and resistant sublines of P388 leukemia. Cancer Res 1979;39:2200–3.[Medline]
  41. Coley HM, Twentyman PR, Workman P. The efflux of anthracyclines in multidrug-resistant cell lines. Biochem Pharmacol 1993;46:1317–26.[CrossRef][Medline]
  42. Xie Y, Ye L, Zhang X, et al. Transport of nerve growth factor encapsulated into liposomes across the blood-brain barrier: in vitro and in vivo studies. J Control Release 2005;105:106–19.[CrossRef][Medline]
  43. Lockman PR, Mumper RJ, Khan MA, Allen DD. Nanoparticle technology for drug delivery across the blood-brain barrier. Drug Dev Ind Pharm 2002;28:1–13.[CrossRef][Medline]
  44. Bally MB, Nayar R, Masin D, Hope MJ, Cullis PR, Mayer LD. Liposomes with entrapped doxorubicin exhibit extended blood residence times. Biochim Biophys Acta 1990;1023:133–9.[Medline]
  45. Abraham SA, Waterhouse DN, Mayer LD, Cullis PR, Madden TD, Bally MB. The liposomal formulation of doxorubicin. Methods Enzymol 2005;391:71–97.[CrossRef][Medline]
  46. Cullis PR, Chonn A, Semple SC. Interactions of liposomes and lipid-based carrier systems with blood proteins: relation to clearance behaviour in vivo. Adv Drug Deliv Rev 1998;32:3–17.[CrossRef][Medline]
  47. Torchilin VP. Immunoliposomes and PEGylated immunoliposomes: possible use for targeted delivery of imaging agents. Immunomethods 1994;4:244–58.[CrossRef][Medline]
  48. Silvius J. Lipid protein interactions. New York: Wiley; 1982.
  49. Regina A, Demeule M, Laplante A, et al. Multidrug resistance in brain tumors: roles of the blood-brain barrier. Cancer Metastasis Rev 2001;20:13–25.[CrossRef][Medline]




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