interleukin-13 receptor–targeted nanovesicles are a ... · nanovesicle drug delivery system for...

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

A.B. Madhankumar,1 Becky Slagle-Webb,1

Akiva Mintz,2 Jonas M. Sheehan,1

and James R. Connor1

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

AbstractThe difficulties associated with treatment of malignantbrain tumors are well documented. For example, localinfiltration of high-grade astrocytomas prevents thecomplete resection of all malignant cells. It is, therefore,critical to develop delivery systems for chemotherapeuticagents that ablate individual cancer cells without causingdiffuse damage to surrounding brain tissue. Here, wedescribe sterically stable human interleukin-13 (IL-13)–conjugated liposomes, which efficiently bind to the braincancer cells that overexpress the IL-13 receptor A2protein. The conjugated liposomes bind to glioblastomamultiforme tissue specimens but not to normal cortex.Conjugating the liposomes with human IL-13 allowsfor specific binding to glioma cells and uptake of theliposomes via endocytosis. Delivering doxorubicin toglioma cells by IL-13–conjugated liposomes results inenhanced cytotoxicity and increased accumulation andretention of drug in the glioma cells compared withdelivery of free drug. The therapeutic potential andtargeting efficacy of the IL-13–conjugated liposomescarrying doxorubicin was tested in vivo using a s.c.glioma tumor mouse model. Animals receiving i.p. injec-tions of IL-13–conjugated liposomes carrying doxorubicinfor 7 weeks had a mean tumor volume of 37 mm3

compared with a mean volume of 192 mm3 in animalsinjected with nontargeted liposomes. These resultsstrongly suggest that IL-13–conjugated liposomes carry-ing cytotoxic agents are a feasible approach for creating ananovesicle drug delivery system for brain tumor therapy.[Mol Cancer Ther 2006;5(12):3162–9]

IntroductionHuman interleukin-13 (IL-13) is a cytokine secreted byactivated T cells that elicits both proinflammatory andanti-inflammatory immune responses (1, 2). IL-13 has twotypes of receptors: IL-13/4R, which is present in normalcells and whose binding is shared with IL-4, and IL-13Ra2,which does not bind IL-4 (3). IL-13Ra2 is associated withhigh-grade astrocytomas more commonly referred to asglioblastoma multiforme (GBM) and is not significantlyexpressed in normal tissue, with the exception of the testes(3–5). A recent study determined that pilocytic astrocyto-mas, the most common astrocytic tumors in children,also overexpress the IL-13Ra2 receptor (6). Thus, theIL-13Ra2 receptor is an excellent potential target fordelivering cytotoxic agents to a variety of devastating braintumors.

A number of attempts to use IL-13Ra2 as a target forhigh-grade astrocytoma therapy have been reported bothin vitro and in vivo. Some of the successful modalities usedinclude IL-13–based cytotoxins (7, 8), IL-13Ra2–targetedviruses (9), and IL-13Ra2 immunotherapy (10, 11). Onesignificant way to enhance the therapeutic index of theanticancer drugs is to specifically deliver these agentsdirectly to the tumor cells through a carrier, therebykeeping them away from healthy cells that are sensitiveto toxic effects of the drugs. Such target-oriented deliverysystems include colloidal delivery systems such as micro-spheres, nanoparticles, liposomes, and micelles. Liposomesare considered a promising drug-delivery technology forbrain tumor therapy (12–16).

Most of the existing liposome-based anticancer therapiesuse nontargeted delivery and display a series of toxic sideeffects to normal cells (17). However, it is thought thattargeted delivery of liposomes should result in increasedaccumulation and retention of liposomes at the tumor site,thus decreasing the systemic toxicity and increasing thetherapeutic ability of liposome-based therapy. Conjugationof functional proteins or monoclonal antibodies to lip-osomes has been extensively explored, primarily to targetspecific cells or tissues (16, 18–20). Our experience informulating colloidal particulate carriers like micelles andmicrospheres (21 – 23) for drug delivery applicationsmotivated us to design IL-13–conjugated liposomes forsite-specific delivery of drugs and diagnostic agents forbrain tumor therapy. Apart from drug targeting, drugtransport to the solid tumors is another area gainingsignificance due to several factors such as multidrugresistance and P-glycoprotein–mediated drug efflux.

In the present work, we developed IL-13–conjugatedliposomes to deliver chemotherapeutic agents specificallyto brain tumors. For our studies, we use doxorubicin as anantineoplastic agent that is widely used for intracranialglioma models in animals (24–27). To establish proof of

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

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

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 UniversityDrive, Hershey, PA 17033-0850. Phone: 717-531-4541;Fax: 717-531-0091. E-mail: [email protected]

Copyright C 2006 American Association for Cancer Research.

doi:10.1158/1535-7163.MCT-06-0480

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our concept, we examined the binding ability of theIL-13–conjugated liposomes in a cell culture model andtheir cytotoxic potential in a glioma cell line. Their ability toovercome P-glycoprotein–mediated expulsion of drug wasalso studied. The cell culture experiments yielded positiveresults. Therefore, the antitumor effect of doxorubicin-encapsulated, IL-13–conjugated liposomes was evaluatedon a s.c. tumor mouse model.

Materials andMethods1,2-Distearoyl-sn -glycero-3-phosphoethanolamine-N -[carboxy(polyethylene-glycol)2000] (ammonium salt),dipalmitoylphosphatidylcholine, cholesterol, and L-a-phos-phatidylethanolamine-N-(lissamine rhodamine B sulfonyl)(ammonium salt) were purchased from Avanti Polar Lipids(Alabaster, AL). Stearylamine was purchased from SigmaChemical (St. Louis, MO). Human U251 and U87 gliomacells were purchased from American Type Tissue CultureCollection (Manassas, VA). Doxorubicin was obtained fromSigma Chemical. Corning 96-well plates were purchasedfrom Corning (Corning, NY); cyclosporine A was fromCalbiochem (La Jolla, CA); and early endosomal antigen 1(EEA1) antibody was from Santa Cruz Biotechnology(Santa Cruz, CA).

PreparationandCharacterizationof IL-13^ConjugatedLiposomes

Sterically stable liposomes were formulated by usingdipalmitoylphosphatidylcholine, cholesterol, 1,2-distearoyl-sn -glycero-3-phosphoethanolamine-N -[carboxy(polye-thylene-glycol)2000], and N-[3-(2-pyridyldithio)-propinyl]stearylamine (molar ratio of 10:5:1.5:1.5) dissolved inmethanol/chloroform mixture (2:1; ref. 28). The liposomeswere subsequently roto-evaporated to obtain a lipid film,which was further dried in a desiccator. For doxorubicinencapsulation or binding studies using HEPES buffer, thelipid film was hydrated in 155 mmol/L ammonium sulfate(pH 5.5) and then sonicated in a bath-type sonicator for15 min. To fluorescently tag the liposomes for the cellularuptake, the liposomes were constructed using 1 mol% offluorescently labeled phospholipid [L-a-phosphatidyletha-nolamine-N-(lissamine rhodamine B sulfonyl) (ammoniumsalt)]. A polycarbonate membrane of gradually decreasingpore size was used to produce small unilamellar vesiclesby extruding through two-stacked, 0.1-Am polycarbonatemembrane and subsequently with 0.05-Am polycarbonatemembrane using a nitrogen pressure–operated extruder(Lipex extruder, Northern Lipids, Inc., Vancouver, BritishColumbia, Canada). All the extrusions were done at anoperating pressure of 800 p.s.i. (5,440 kPa). The liposomeswere then purified and sterilized by passing throughSephadex G25M column. The liposome concentration wasdetermined by phosphate assay (29). The size distributionof the liposomes was determined by dynamic lightscattering, which was conducted using an ALV/DLS/SLS-5022F compact goniometer system (ALV, Langen,Germany), which was confirmed by transmission electronmicroscopy using uranyl acetate as the staining agent.

Human IL-13 gene, which was isolated from the totalRNA (BD Biosciences, Mountain View, CA) from humantestis by reverse transcription-PCR, which was cloned intothe TOPO vector (Invitrogen, Carlsbad, CA), expressed inEscherichia coli as His-tagged protein and purified by nickelaffinity binding. Conjugation of IL-13 to liposomes wasdone according to the method reported by Singh et al. (28).A heterobifunctional reagent, N-succinimidyl-3(2-pyridyl-dithio)propionate, was used to introduce pyridyl disulfidegroups to the IL-13 molecule (28). Briefly, 10 mol of N-succinimidyl-3(2-pyridyldithio)propionate was dissolvedin methanol and then this solution was reacted with1 mol of IL-13 in PBS for 24 h at 4jC. The unreactedN-succinimidyl-3(2-pyridyldithio)propionate was removedby dialysis against PBS (molecular weight cutoff, 10,000).The dialysate was reduced with DTT (20 mmol/L finalconcentration), and the unreacted excess DTT was removedby gel filtration through a Sephadex G25M column.Thiolation of IL-13 was verified by the presence of freesulfhydryl groups, which were estimated by Ellman’smethod (30) according to Ellman’s reagent protocol (Pierce,Rockford, IL).

Thiolated IL-13 protein was slowly added to a 5-mLbeaker containing the liposomes and a magnetic stirringbar and incubated overnight with slow stirring at 4jC.The conjugated liposomes were separated by ultracentri-fugation at 40,000 rpm. The IL-13 to phospholipid moleratio was maintained at 1:700. After conjugation, thepresence of IL-13 protein on the liposomes were verifiedby Coomassie (Bradford; Pierce) protein binding assay.The lipid content of the liposome was measured by phos-phorus estimation according to the method of Morrison (31).

To add transferrin to the liposomes, commerciallyavailable bovine Tf (Sigma Chemical) was conjugated toIL-13 liposomes by a similar method to that described forIL-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 lip-osomes using a phospholipid to Tf mole ratio of 1:700using the same methods and conditions as that for IL-13conjugation.

Method of Encapsulation of Doxorubicin in theLiposomes

Doxorubicin was encapsulated into the liposomes byammonium sulfate gradient method (32). The liposomeswere hydrated with ammonium sulfate (pH 5.5; 155 mmol/L)using a bath-type sonicator. The liposomes were thenextruded as before. The concentration of phospholipidwas maintained at 10 mmol/L. The external buffer wasexchanged by passing the liposomes through SephadexG-25M column and eluting them with 123 mmol/L sodiumcitrate (pH 5.5). Then, the liposomes were incubated withdoxorubicin (0.2 mg doxorubicin per milligram of phos-pholipid) for 1 h at 65jC. In all our preparations, the drugto lipid weight ratio was maintained as 1:5. Unencapsu-lated doxorubicin was removed by passing the liposomesthrough Sephadex G25M column and exchanging themwith PBS.

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Doxorubicin Leakage StudyThe leakage of doxorubicin from the IL-13–conjugated

liposomes and the unconjugated liposomes was deter-mined by suspending 10 AL of doxorubicin-containingliposomes in 0.5 mL of human serum or dialyzed against alarge volume of PBS. Both experiments were done at 37jCfor increasing time intervals. Both serum and PBS mediawere evaluated to compare shelf life (PBS) and in vivostability for delivery (serum). To determine the amounts ofdoxorubicin that may have been released from the lip-osomes, the serum samples were centrifuged at 40,000 rpmand the supernatants were analyzed for doxorubicin bymeasuring the absorbance at 492 nm. For the PBS studies,the dialysate was collected and assayed for doxorubicin.

Uptake of IL-13^ Conjugated Liposomes in Normaland Glioma Cells

Uptake of the IL-13–conjugated liposomes on gliomacells was done to investigate the ability of the glioma cellsto internalize the liposomes. Both U251 and U87 gliomacells (10,000 cells each) were cultivated on a chamber slidefor 24 h. IL-13–conjugated, rhodamine-labeled liposomeswere added for 120 min at 37jC. Human umbilical veinendothelial cells and SVGp12 glial cells (purchased fromAmerican Type Culture Collection) served as controls. TheSVG p12 cell lines are human fetal glial cells from brainmaterial, which are transfected with DNA from an ori-mutant of SV40. The cells were washed thrice with PBS toend the exposure to liposomes and then viewed with aconfocal microscope. The cells were stained with 4¶,6-diamidino-2-phenylindole to visualize the nuclei.

To determine if the uptake of the liposomes is involvedthe endosomal system, U251 glioma cells were cultured onchamber slides as described above and the cells werepermeabilized and blocked for 30 min in 0.1% bovineserum albumin and PBS (blocking buffer). The cells weretreated with rhodamine-labeled IL-13–conjugated lipo-somes and then stained with polyclonal EEA1 antibody(1:15) for 30 min. The cells were then washed thrice withPBS and counterstained with FITC–anti-goat antibody(1:75) for 30 min and observed under fluorescent micro-scope. The images were captured using a digital camera.

Flow CytometryFlow cytometry was used to measure total intracellular

doxorubicin fluorescence (33). In this report, we refer tofluorescence intensity as intracellular drug content. Cells(1 � 106) were exposed to 20 Amol/L of drug as (a) freedoxorubicin; (b) doxorubicin-encapsulated, IL-13–conju-gated liposomes; and (c) doxorubicin-encapsulated uncon-jugated liposomes for 2 h. All drug treatments andposttreatment incubations were done in complete growthmedium. The cells were washed to remove any freeadherent doxorubicin using PBS and centrifuged. Cellswere released from tissue culture dishes with 0.05%trypsin/0.02% EDTA followed by PBS washing (centrifu-gation, 5 min, 500 � g) and resuspended in PBS for flowcytometry assay. The intracellular accumulation of inher-ently fluorescent doxorubicin was evaluated using afluorescence activated cell analyzer. A single 15-mW argon

ion laser beam (488 nm) was used to excite the fluorescenceof doxorubicin. A total of 10,000 cells were analyzed foreach histogram. Experiments were repeated thrice and thefluorescence intensities of doxorubicin were expressed inarbitrary units.

Binding to Human BrainTumor SectionsTo show the potential clinical application of the conju-

gated liposomes, we obtained GBM and pilocytic astrocy-toma brain tumor sections and exposed them to therhodamine-labeled IL-13 liposomes. Brain tumor sampleswere obtained from patients undergoing surgical decom-pression at Penn State University Hershey Medical Center.All studies involving human specimens were approved bythe respective Human Subjects Protection Office at thePenn State College of Medicine (protocol no. 96-123EP).Serial tissue sections were generated (10 Am) on a cryostat,thaw mounted on chromalum-coated slides, and stored at�70jC until analyzed. The sections were then blocked withnormal goat serum (10%) and then exposed to rhodamine-labeled, IL-13–conjugated liposomes for 1 h at 37jC. Then,sections were washed thrice with PBS before observingthem via fluorescence microscopy. To test the hypothesisthat the IL-13–conjugated liposomes interacted with the IL-13 receptor on the GBM tumors, some of the GBM sectionswere blocked with 1 mg/mL concentration of IL-13Ra2receptor antibody and followed by exposure to rhodamine-labeled, IL-13–conjugated liposomes. The sections werethen washed with PBS and observed under a fluorescencemicroscope.

Effect of P-Glycoprotein Inhibitor on the Internaliza-tion of IL-13^ Conjugated Liposomes in the Glioma Cells

About 50,000 U251 glioma cells were plated in a smallPetri dish and were exposed to either IL-13–conjugatedliposomes carrying 20 Amol/L of doxorubicin or to thesame concentration of free doxorubicin for 2 h. The cells ineach condition were either treated or not with cyclosporineA, a P-glycoprotein inhibitor (5 Ag/mL), for 30 min beforeaddition of the liposomes. After 2-h incubation, cells werewashed with PBS, removed with versene, and subjected toflow cytometry.

Cytotoxicity Assay with Doxorubicin-Encapsulated,Ligand-Targeted Liposomes

In our experiments, we used doxorubicin-encapsulatedliposomes, which are unconjugated, conjugated with IL-13,or double conjugated with IL-13 and Tf, to determine theircytotoxic potential. Cytotoxicity was measured after addingserially diluted doxorubicin-encapsulated liposomes toU251 glioma cells plated in 96-well cell culture plates at aconcentration of 5 � 103 per well. Cell survival wasdetermined after 48 h by 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H -tetrazoli-um/phenazine methosulfate assay. Cells treated with highconcentrations of cycloheximide served as the backgroundfor the assay (34, 35).

In vivo Therapeutic Efficacy ofTargeted LiposomesTo test the in vivo efficacy of the targeted liposomal

system, adult female athymic nude mice were implantedin the flank s.c. with U251 glioma cells. Exponentially

IL-13 Receptor–Targeted Nanovesicles for GBM Therapy3164




growing cells were harvested and 15 � 106 cells per mousewere s.c. injected. After 2 weeks, a tumor of volume 14 to30 mm3 was observed. At that time, the mice were dividedinto five different groups of six mice in each group. Onegroup of mice was injected with IL-13–conjugated lipo-somes carrying doxorubicin at a dosage of 15 mg/kg ofbody weight. A second group was injected with the sameamount of liposomes carrying 15 mg/kg body weight ofdoxorubicin, but these liposomes were unconjugated. Athird group received injections of IL-13–conjugated lip-osomes but with a lower dosage of doxorubicin (7.5 mg/kgof body weight). The fourth group of mice were untreatedand received injections of 0.1 mol/L PBS as a control. Afifth group of mice were injected with unconjugatedliposomes carrying no drug as an additional control. Allthe drugs were administered i.p. weekly. The injectionswere given opposite the side of the s.c. tumor. The tumorsize, health, and survival of the mice were monitoredweekly by an investigator (B.W.) blinded as to the groupsof mice. These experiments were approved by the Penn-sylvania State University Institutional Animal Care andUse Committees.

ResultsLiposome Composition and Particle SizeThe particle size of the liposome as confirmed by laser

particle size analyzer and transmission electron microscopywas found to be in the range of 50 to 150 nm with a meansize of 104 nm. The polydispersity index for various

batches of nanovesicles consistently lies in the range of0.2 to 0.4. After conjugation and purification, the concen-tration of the phospholipids in the liposome was 21.8 Agof phospholipids per microliter and the concentration ofIL-13 conjugated on the liposomes after doxorubicinencapsulation was 3.46 � 10�7 Amol of IL-13 per microgramof phospholipids. The final concentration of doxorubicin is0.18 Ag/Ag of phospholipid.

We also observed the effect of temperature on encapsu-lation efficiency of doxorubicin to be maximum (90%) at65jC when compared with lower temperatures, 25jC and40jC, where the encapsulation efficiencies are 45% and72%, respectively. The T1/2 for doxorubicin leakage fromIL-13 liposome at 37jC in PBS was 25 days, whereas withunconjugated liposomes, the T1/2 is f45 days. Thus, theIL-13–conjugated liposomes were not substantially leakyduring the experimental period, because our experimentswere done within 2 weeks of preparation. We did notobserve any significant leakage of doxorubicin from theliposomes that were incubated in human serum at 37jC forat least 1 week.

Binding to Glioma CellsFor IL-13 receptor–targeted liposomes to be considered

for clinical use, it is necessary to show that glioma cells takeup the liposomes. Figure 1 shows the uptake of theliposomes in both U87 and U251 glioma cell lines, whereasnormal cells like human umbilical vein endothelial cellsand the immortalized glial cell line SVGp12, which do notoverexpress IL-13 receptor, had no detectable uptake overthe same exposure time (36).

Figure 1. Uptake of IL-13–conjugatedrhodamine-labeled liposomes in human celllines using rhodamine-labeled liposomes.Uptake is clearly visible in the U251 andU87 glioma cells. Red, rhodamine staining;blue, 4¶,6-diamidino-2-phenylindole nuclearstaining. SVGp12 and human umbilical veinendothelial cells (HUVEC ) served as negativecontrols. The cells were exposed to theliposomes for 2 h.





Intracellular Accumulation and Retention of Doxo-rubicin in U251 Glioma Cells

The uptake and accumulation of the IL-13–conjugatedliposomes were analyzed using flow cytometry andfluorescent microscopy. The IL-13–conjugated liposomesenter early endosomes as shown by colocalization withEEA1 (Fig. 2). The relative accumulation of the doxorubicinin U251 cells depending on the mode of delivery wasshown by flow cytometry (Fig. 3). The ability to showdoxorubicin in cells by fluorescence-activated cell sorting(FACS) analysis takes advantage of the intrinsic fluores-cence of doxorubicin when excited at 488 nm (37, 38). Theflow cytometry analysis showed a right shift in thecurve, indicating an increase in the cell fluorescence inU251 glioma cells after exposure to free doxorubicin orliposomal doxorubicin. The right shift is greater withIL-13–conjugated liposomal doxorubicin than with non-conjugated liposomes (Fig. 3A). It is well known thatdrug accumulation in the cancer cells is decreased byP-glycoprotein activity (39–41). When doxorubicin is deli-vered by IL-13–conjugated liposomes, the intrinsic fluo-rescence of the doxorubicin accumulated or retainedintracellularly in U251 glioma cells is much higher thanthat seen in cells exposed to free doxorubicin (Fig. 3B).Indeed, the level of doxorubicin detected in the cells follow-ing delivery via liposome was even greater than that seenin cells treated with free doxorubicin, which were also ex-posed to cyclosporine A, a P-glycoprotein inhibitor (Fig. 3B).

Exposure of GliomaTumors to LiposomesRepresentative samples of GBM, pilocytic astrocytomas,

and normal human cortex exposed to IL-13–conjugatedliposomes tagged with rhodamine are shown in Fig. 4.There was a much greater affinity of the GBM and pilocyticastrocytoma samples for the IL-13–conjugated liposomesthan the medulloblastoma or normal human cortexsamples. The specificity of this association of IL-13 –conjugated liposomes to the IL-13 receptor was shown byexposing the tumor sections to IL-13 receptor antibodyfollowed by IL-13 –conjugated vesicles. This approachresulted in a decrease in the binding of IL-13–conjugated,rhodamine-labeled liposomes.

CytotoxicityAssay with Ligand-Targeted LiposomesThe cytotoxicity of doxorubicin on U251 glioma cells

encapsulated in IL-13–conjugated liposomes versus un-conjugated liposomes was compared and the results are

Figure 2. Colocalization of IL-13–conjugated, rhodamine-labeled liposomes with EEA1 using U251 glioma cells. The cells were exposed to theliposomes and subsequently with EEA1 for 40 min. A, IL-13 liposomal uptake (red). B, cells were exposed to EEA1 and then labeled with FITC-conjugatedanti-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. Flow cytometric analysis of doxorubicin accumulation inU251 glioma cells. A, retention of doxorubicin in U251 glioma cells afterexposure to doxorubicin in unconjugated liposomes (LIPDXR ) anddoxorubicin in IL-13–conjugated liposomes (IL13LIPDXR ). Encapsulatingdoxorubicin in IL-13–conjugated liposomes results in an increaseaccumulation of doxorubicin over 2 h of exposure. B, U251 glioma cellsexpress P-glycoproteins and are a good model for examining multidrugresistance. Therefore, we compared the accumulation of free doxorubicinin the presence (DXR-Cyclo ) or absence (DXR ) of cyclosporine A, aP-glycoprotein inhibitor. Cells were exposed to doxorubicin for 2 h. Thepresence of cyclosporine A resulted in retention of doxorubicin to levelsthat were similar to the IL-13 liposome–delivered doxorubicin. Histogramsare data from one representative experiment. The experiments were donethrice for each condition. Control in each condition are cells alone with notreatment.





shown in Fig. 5. Because we were also evaluating thepossibility of using liposomes doubly conjugated with Tfand IL-13, these liposomes were also included in thisexperiment. The concentration of liposomes, which wereadded to each of the cell cultures, was identical; eachliposome carried equal amounts of doxorubicin. At thelowest concentration, 150 ng/mL of liposomal doxorubicin,IL-13–conjugated liposomes were 31.7% more cytotoxicthan unconjugated liposomes (P < 0.001). The cytotoxicityof the doubly conjugated (IL-13, Tf) liposomes was similarto the IL-13–conjugated liposomes (35.3%). With increasingconcentration, the liposomal doxorubicin cytotoxicityincreases at a faster rate than the cytotoxicity associatedwith the unconjugated liposomes.

In vivo Anticancer Therapeutic EfficacyThe tumors in the control mice grew from 14 to 570 mm3

in 7 weeks, whereas the tumor growth rate is much lowerin animals that received doxorubicin-carrying liposomes.The most effective approach at reducing the tumors was IL-13–conjugated liposomes carrying doxorubicin (15 mg/kgof body weight). In this group, the tumor volume decreasedby 69% over the first 2 weeks after injections (Fig. 6). Theonly other group to show an initial decrease in tumor size(52%) was the one receiving injections of unconjugatedliposomes carrying doxorubicin. The group receiving thehighest dose of the conjugated liposomes and doxorubicinhad a tumor volume of only 37 mm3 or <10% of theuntreated group after 7 weeks (termination of the exper-iment). Animals receiving the same dose of unconjugatedliposomes had a tumor volume of 192 mm3 in 7 weeks,5-fold more than the animals receiving the same concen-tration of doxorubicin in targeted liposomes and 22%higher than animals receiving the lowest dose of doxoru-bicin in conjugated liposomes (Fig. 6). In the group thatreceived only liposomes (untargeted and not containingdoxorubicin), the tumor volume did not decrease appre-ciably and at the end of 7 weeks had an average volume of452 mm3. During the course of these studies, only one

animal died. This animal was in the high doxorubicingroup (conjugated liposomes) and the death seemedrelated to an injection artifact. No animals died in theother groups.

DiscussionPreviously, IL-13 receptors have been identified as apotential target on high-grade astrocytomas, but theoutcomes have been mixed. Here, we show an alternativeapproach of using IL-13–conjugated liposomes to selec-tively target and deliver the cytotoxin doxorubicin to tumorcells 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 arelatively extended half-life (44). In addition, the antitumor

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

Figure 4. Affinity of rhodamine-labeled IL-13–conjugated liposomes for brain tissuesamples. A number of brain tissue samples(normal and tumor) were examined foraffinity to the IL-13–conjugated liposomes.The results show highest affinities of theliposomes from GBM and relatively less, butstill detectable, affinities from pilocytic as-trocytoma. No binding is observed to medul-loblastoma or to the normal human cortex.To show that the IL-13–conjugated lip-osomes have affinity for the IL-13 receptor,a GBM tissue section (GBM#15 ) was firstexposed to IL-13 receptor antibody and thenexposed to the liposomes. This approachresulted in a decrease in binding of the GBMwith the liposomes.





activity of the liposomal doxorubicin is sensitive to vesiclesize, and the liposomes in this size range can readily releasetheir contents within the cells (45), which is consistent withour observations in this study.

Our liposome system is composed of polyethylene glycollipids 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 portionrather than on the surface of the polyethylene glycolmoiety. Our data indicate that the liposomes constructed inthis manner maintain their targeting property, maintain anability to effectively encapsulate and retain the drugfollowing covalent attachment of IL-13, and are still ableto bind the target IL-13Ra2 on glioma cells. Moreover, ourliposomes are relatively stable and, unlike egg-phosphati-dylcholine/cholesterol liposomes, after drug encapsula-tion, they are not leaky in serum or buffer at physiologictemperatures. The liposomes configured in our study onlybecame leaky at a temperature well above the transitiontemperature of dipalmitoylphosphatidylcholine (48).

Our study showed effective binding of IL-13–conjugatedliposomes to the malignant glioma cells and the clinicalspecimens of brain tumors in situ . We provided evidencefor an affinity to high-grade astrocytoma (GBM) as well as

a low-grade pilocytic astrocytoma. These observations areconsistent with a recent report where the presence of IL-13Ra2 was shown on these tumors (6). The uptake studiesshowed that the liposomes were found in early endosomes,which is consistent with receptor-mediated uptake. Thelack of affinity of the liposomes for the normal humancortex or the human umbilical vein endothelial cells isconsistent with the absence of detectable IL-13Ra2 receptor.

The cytotoxicity experiments and in vivo experimentsrevealed that the IL-13–conjugated liposomes were supe-rior to the unconjugated liposomes at killing the tumorcells. Most brain tumors express P-glycoprotein, whichconfers drug resistance to glioma cells (49). Our resultsshowed that the liposome-delivered doxorubicin was notexpelled by P-glycoprotein from the cell, unlike theunencapsulated doxorubicin. Therefore, the explanationfor the enhanced cytotoxicity with the IL-13–targetedliposomes is that doxorubicin delivered by these liposomesresults in increased accumulation and retention in gliomacells. The demonstration that liposome-delivered doxoru-bicin can avoid expulsion from tumor cells in a cell culturemodel while having greater efficacy in the in vivo modelstrongly supports the notion that liposomal delivery is aviable option for brain tumors in vivo.

A critical component of drug delivery systems is theirability to target the tumors without adverse effect to thenormal healthy tissues and to transport therapeutic agentsinto the tumors overcoming the P-glycoprotein–mediateddrug resistance. In our in vivo model, we could clearlyobserve higher therapeutic efficacy of the IL-13–conjugatedliposomes where the tumor volume was reduced by 68%in 3 weeks, whereas in the unconjugated liposomes thetumor volume was only reduced by 50% over 3 weeks. Thedifference in final volume (7 weeks) between conjugatedand nonconjugated liposomes (over 500%) is compellingevidence that IL-13–conjugated liposomes carrying doxo-rubicin are much more efficacious than untargeted lip-osomes carrying the same amount of doxorubicin. The cellculture data suggest that the greater efficacy of the targetedliposomes is a combination of the receptor targeting natureof the liposomes and the ability of the targeted liposomesto overcome the P-glycoprotein–mediated drug efflux bythe tumor. Thus, IL-13 receptor– targeted nanovesiclesrepresent a viable approach where the liposomes of particlesize ranging from 50 to 150 nm can be used to deliverchemotherapeutic agents to brain tumor cells and may be aviable option for i.v. drug delivery applications across theblood-brain barrier.

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Figure 6. The therapeutic efficacy of the IL-13 receptor– targetedliposomes carrying doxorubicin was tested in a s.c. glioma tumor model innude mice. Mice were given i.p. injections weekly. A, mice receivingtargeted liposomes with doxorubicin (DXR) had a greater reduction intumor size in the first 2 wks compared with the animals receiving the sameconcentration of unconjugated liposomes and doxorubicin. The tumors inall 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 liposomeswithout drug (LIP without DXR ). Points , mean tumor volume; bars , SE.Error bars on the LIP (DXR ) 15 mg/kg group are contained within thesymbol for this group.





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2006;5:3162-3169. Mol Cancer Ther A.B. Madhankumar, Becky Slagle-Webb, Akiva Mintz, et al. therapy for glioblastoma multiforme

targeted nanovesicles are a potential−Interleukin-13 receptor

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