integrinb6-targeted immunoliposomes mediate tumor-speciﬁc ... · for the cellular uptake studies,...

Cancer Therapy: Preclinical

Integrinb6-Targeted Immunoliposomes MediateTumor-Specific Drug Delivery and EnhanceTherapeutic Efficacy in Colon CarcinomaBenjia Liang1,2, Muhammad Shahbaz1, Yang Wang3, Huijie Gao1,2, Ruliang Fang1,Zhengchuan Niu1,2, Song Liu1,2, Ben Wang1,2, Qi Sun1,2,Weibo Niu1, Enyu Liu1,Jiayong Wang1, and Jun Niu1

Abstract

Purpose: Adjuvant chemotherapy is one of the significanttreatments for colon cancer in clinic. However, it does not achievethe desired therapeutic efficacy, largely due to chemotherapeuticresistance. Integrinb6 (ITGB6) is expressed in malignant colonicepithelia, but not in normal epithelia, and is associated with theprogression, metastasis, and chemotherapeutic resistance ofcolon cancer. Accordingly, it is necessary to design therapeuticapproaches for efficient and targeted drug delivery into ITGB6-positive cancer cells to improve chemotherapeutic efficacy incolon cancer.

Experimental Design: PEGylated liposomes were employed todesign ITGB6-targeted immunoliposomes, which have ITGB6monoclonal antibodies (mAbs) conjugated. We evaluated theITGB6-targeted immunoliposomes internalization into coloncancer cells and examined 5-fluorouracil (5-FU)–induced cellularapoptosis produced by ITGB6-targeted immunoliposomesþ5-FU. In addition, the biodistribution and antitumor efficiency ofITGB6-targeted immunoliposomes were observed in vivo.

Results: ITGB6-targeted immunoliposomes enhanced cellularinternalization in ITGB6-positive colon cancer cells comparedwith liposomes. Furthermore, the ITGB6-targeted immunolipo-some internalization was dependent on the ITGB6 expressionlevel on cellular surface. ITGB6-targeted immunoliposomesdecreased the 5-FU IC50 more than 90% in HT-29 and SW480b6cells relative to liposomes. Moreover, when loaded with 5-FU,ITGB6-targeted immunoliposomes produced an approximately1.5-fold higher 5-FU–induced cellular apoptosis rate than lipo-somes. In vivo, the therapeutic activity of ITGB6-targetedimmunoliposomesþ5-FU was significantly superior, resulting in25% to 35% reduction of tumor weight compared with 5-FU orliposomesþ5-FU.

Conclusions: ITGB6-targeted immunoliposomes provide ahighly efficient approach for targeted drug delivery in coloncancer and thus offer the potential of a novel and promisinganticancer strategy for clinical therapy. Clin Cancer Res; 21(5);1183–95. �2014 AACR.

IntroductionColon cancer is the third most common cancer and the fourth

leading cause of cancer-related deaths worldwide (1). Althoughsurgery remains the preferred treatment, 5-fluorouracil (5-FU)–based adjuvant chemotherapy is the conventional care for stage III(lymph node–positive) patients and can reducemortality by 25%compared with surgery alone (2). However, because the responserate of chemotherapy is only 10% to 20%, the treatment ofadvanced and metastatic cases remains a challenging problem(3). Therefore, it is urgent to explore novel therapeutic strategiesfor colon cancer treatment to overcome chemotherapeutic drugresistance and to improve chemotherapeutic efficacy.

The targeted delivery of anticancer drugs to tumors has beenbroadly recognized as an important method for improving che-motherapeutic efficacy and attenuating chemotherapeutic sideeffects. Sterically stabilized liposomes with a polymeric PEGcoating, which have prolonged circulation times, lower reticulo-endothelial system (RES) uptake andhigher tumor accumulation,were thought to be an ideal drug delivery system (4, 5). However,these liposomes passively interact with tumor cells, in vitro or invivo, resulting in nonspecific drug release that leads to the eventualdiffusion of the drugs into somenormal tissues or cells rather thantumors (6).

Immunoliposomes, which are conjugated with monoclonalantibodies (mAb), are capable of both drug delivery andmolecular targeting (7, 8). By combining the specific targetingproperties of mAbs and drug delivery advantages of liposomes,immunoliposomes are a promising approach for the targeteddelivery of anticancer drugs to tumors (9). To date, varioustumor-associated antigens have been validated as targets forantibody-based immunoliposomes in cancer therapeutics.Immunoliposomes targeting CD30, HER2/neu, EGFR, andVEGFR, which are expressed on various tumor cell types, havebeen developed and thoroughly characterized (10–13). On thebasis of encouraging preclinical data and advances in large-scale production processes, EGFR-specific immunoliposomesare already being used in clinical trials (14).

1Department of Hepatobiliary Surgery, Qilu Hospital, Shandong Uni-versity, Jinan, Shandong, P.R. China. 2Key Laboratory of Cardiovascu-lar Remodeling and Function Research, ChineseMinistry of Educationand Public Health, Jinan, Shandong, P.R. China. 3School of Pharma-ceutical Sciences, Shandong University, Jinan, Shandong, P.R. China.

Corresponding Author: Jun Niu, Department of Hepatobiliary Surgery, QiluHospital, Shandong University, 44 Wenhua Xi Road, Jinan 250012, Shandong,China. Phone:/Fax: 86 531 82166651; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-1194

�2014 American Association for Cancer Research.

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Published OnlineFirst December 30, 2014; DOI: 10.1158/1078-0432.CCR-14-1194

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Integrinb6 (ITGB6) is a subtype of integrin that is expressedexclusively on the surfaces of epithelial cells and is a receptor forextracellular matrix proteins (15). ITGB6 expression is upregu-lated during embryogenesis, oncogenesis, and epithelial repair,whereas it is generally undetectable in healthy epithelial tissues(16). In colon cancer, ITGB6 is specifically expressed in tumortissues and is rarely present in tissues adjacent to the tumor (17).In addition, we previously reported that ITGB6 was associatedwith colon cancer pathology, malignancy, and TNM stage andcould act as a prognostic indicator in aggressive colon carcinomas(18). Our research previously confirmed that ITGB6 contributedto chemotherapeutic resistance in colon cancer; ITGB6 protectedcolon cancer cells from 5-FU–induced growth inhibition andapoptosis (19). Unsurprisingly, the exclusive expression of ITGB6and its influential effects in colon cancer make it a novel thera-peutic target for colon cancer treatment.

ITGB6haspreviously been employed as a clinical biomarker forearly cancer detection (20, 21). Moreover, ITGB6 signaling axis-blocking agents have also been designed to exploit this receptor asa therapeutic target for cancer treatment (22, 23). However,research concerning ITGB6-targeted drug delivery for colon cancerchemotherapy has not been reported. In the current study, wedescribe the design, preparation, and characterization of ITGB6-targeted immunoliposomes, and explore their antitumor efficien-cy against colon cancer in vitro and in tumor xenograft modelsusing 5-FU–loaded ITGB6-targeted immunoliposomes.

Materials and MethodsMaterials

Hydrogenated soy phosphatidylcholine (HSPC) and choles-terol were purchased from Nippon Fine Chemical. Distearylpho-sphatidylethanolamine (DSPE)-mPEG (2000) and DSPE-PEG(2000)-NH2 were purchased from Avanti Polar Lipids. Thehomobifunctional crosslinker bis (sulfosuccinimidyl) suberate(BS3) was from ProteoChem. 5-FU, organic solvents, and otherreagent purity chemicals were from Sigma. The cytochrome Capoptosis assay kit and caspase fluorometric assay kit wasobtained from Biovision. Phosphorylated ERK1/2 (p-ERK1/2),caspase-3/9, cleaved PARP and GAPDH antibodies were pur-chased from Santa Cruz Biotechnology, Inc., and cleaved cas-pase-3 and b-actin antibodies were from Cell Signaling Technol-ogy, Inc. The monoclonal antibody that detects the extracellulardomain of human ITGB6 (E7P6) was prepared and provided as agift from Michael Agrez (The University of Newcastle, Newcastle,Australia). The Annexin V–FITC/propidium iodide (PI) cell apo-

ptosis assay kit was purchased from Merck Millipore. The cellcounting kit-8 (CCK-8) was from Dojindo Molecular Technolo-gies. The In Situ Cell Death Detection Kit was obtained fromRoche.

Cell linesHuman colon cancer cell lines, SW480 and HT-29, were

obtained from the ATCC. SW480 colon cancer cells lack consti-tutive ITGB6 expression. SW480b6 cells that were stably trans-fected with pcDNA1neo constructs containing the ITGB6 geneand SW480 mock cells expressing only plasmid were prepared aspreviously described (24). HT-29 cells constantly express ITGB6.HT-29 cells with siRNA suppressed ITGB6 expression were alsoprepared as reportedpreviously (25). The cellsweremaintained asmonolayers inmedium comprisingDMEM(Hyclone) containing10% heat inactivated FCS (Gibco) and supplemented with 20mmol/L HEPES, 100 IU/mL penicillin, and 100 mg/mL strepto-mycin. The cells were incubated in 37�C, 5% CO2, and saturatedhumidity.

Liposome preparationThe nontargeted liposomes that were to be loaded with 5-FU

were composed of HSPC, cholesterol, and DSPE-PEG2000 at amolar ratio of 2:1:0.1. The liposomes were prepared using pHgradients combined with the reverse-phase evaporation method.Briefly, lipids were dissolved in a mixture of chloroform andmethanol (9:1 v/v). After addition of 0.3 mol/L sodium citratebuffer (SCB, pH ¼ 4.0) of 5-FU containing DSPE-PEG2000, themixture in a ratio 4:1 (v/v) between organic and aqueous phasewas sonicated at room temperature, and the chloroform andmethanol were evaporated using a rotary evaporator. The pH wasthen adjusted to 7.0 using 0.4 mol/L sodium phosphate dibasicbuffer. The liposomes were shaken in a vortex to form an aqueoussuspension and were subsequently extruded 10 times throughpolycarbonate filters with a defined pore size of 400–100 nm. Theliposomes encapsulating 5-FU were separated from free 5-FUusing a Sephadex G-50 column. The concentrations of liposomaland free 5-FUwere determined by spectrophotometry. The encap-sulation efficiency (EE%) of drugs was calculated from the equa-tion:

EE% ¼ Wencapsulated

Wencapsulated þWfree� 100%

ITGB6-targeted immunoliposomes were composed of HSPC/Chol/DSPE-PEG2000/DSPE-PEG2000-NH2 at a molar ratio of2:1:0.08:0.02. These liposomes were prepared and loaded with5-FU as described above. Then ITGB6-specific antibody E7P6 wascoupled to liposomes using the previously reportedmethod (26).The cross-linker BS3 was applied to activate the DSPE-PEG2000-NH2 in the liposomes, which were then conjugated to the aminogroups of the ITGB6 antibody. Briefly, BS3 was added to theliposomes at afinal concentration of 5mmol/L andwas incubatedfor 1 hour at room temperature. The excess unreacted cross-linkerwas removed via ultrafiltration using an Amicon Ultra-0.5 3kD(Millipore). The activated liposomes were collected and incubat-ed with ITGB6 antibody for 2 hours at room temperature. Toquench the reaction, 50 mmol/L Tris was added at room tem-perature for 15 minutes. The immunoliposomes were separatedfrom the unconjugated ITGB6 antibody using Sepharose CL-4Bcolumns. For the cellular uptake studies, liposomes containingcoumarin-6 were prepared by the same method.

Translational Relevance

The targeted delivery of anticancer drugs into tumors is animportant factor for improving the therapeutic efficacy ofcancer treatments. By combining specific targeting and drugdelivery, immunoliposomes are a promising approach for thetargeted delivery of anticancer drugs to tumors. Our group hasconfirmed that integrinb6 (ITGB6) is associated with theinvasion, metastasis, and chemotherapeutic resistance ofcolon cancer. In this study, we describe preclinical data thatsupport the therapeutic efficacy of ITGB6-targeted immuno-liposomes against colon cancer.

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Clin Cancer Res; 21(5) March 1, 2015 Clinical Cancer Research1184




For the in vitro release studies, 1 mL of 5-FU liposomes wasplaced into a dialysis bag and dialyzed against phosphate buffer(200 mL, pH 7.4). The medium was stirred at 37�C. At prede-termined time intervals, 2 mL of the medium was removed andreplaced with an equal volume of phosphate buffer. The released5-FU was quantified using spectrophotometry. Drug release stud-ies in the presence of serum were conducted in a similar manner.In brief, 200 mL of the liposomes was mixed with 800 mL of FBSand was then dialyzed against buffer at pH 7.4 at 37�C. Sampleswere removed at specified timepoints and the concentrations of 5-FU were determined. Release of 5-FU from immunoliposomes atvarious values of pHwas investigated with buffers (pH¼ 6.5, 7.5,and 8.5) using the same method.

Characterization of liposomesThe morphologies of liposomes and ITGB6-targeted immuno-

liposomes were examined by transmission electron microscopy(TEM; JEM-100CX II, Japan). The samples were placed onto acopper grid and air-dried, followed by negative staining with adrop of 2% phosphotungstate solution.

Themean particle size and distribution of liposomes, as well aszeta-potential values, were determined by Malvern ZetasizerNano ZS90.

Cellular uptake and internalization studiesThe cellular uptake of liposomes was examined using a fluo-

rescence microscope. Cells were seeded on chambered coverslipsin 24-well culture plates (2� 104 cells/well) andwere cultured for24 hours. Then, coumarin-6 loaded liposomes, ITGB6-targetedimmunoliposomes, or free coumarin-6were added to eachwell ata final coumarin-6 concentration of 2 mg/mL. Cells cultured withonly medium served as blank controls. After a 1-hour incubation,the medium was removed and the cells were washed three timeswithPBS. Then, the cellswerefixedwith 4%paraformaldehyde for15 minutes. After being washed twice with PBS, the cell nucleiwere stained with DAPI for 10 minutes. The fluorescence imageswere analyzed using a fluorescence microscope (OLYMPUS IX81,Japan). The cellular internalization of the various liposomes wasalso analyzed using flow cytometry. HT-29 and SW480b6 cellswere seeded onto 6-well plates (1� 105 cells/well). The cells werecultured and treated as described above. The cells were trypsinizedandwashed three timeswithPBS. Then, the cellswere collected viacentrifugation and were resuspended in 500 mL of PBS. Cellularuptake was analyzed by flow cytometry.

Cytotoxicity studiesThe cytotoxicity studies were performed using the CCK-8 assay.

Cells were seeded in 96-well cell culture plates (1� 104 cells/well)and incubated for 24 hours. Then, the medium was exchangedwith 100 mL fresh DMEM containing free 5-FU, liposomesþ5-FU,or immunoliposomesþ5-FU [(5-FU)¼ 0.2, 1, 5, 25, 125 mg/mL).Cells treatedwith serum-freemediumwere used as a control. Afterincubation for predetermined times, the cell viability was deter-mined using a CCK-8 assay kit according to the manufacturer'sinstructions. After adding 10 mL CCK-8 to eachwell, followed by a2-hour incubation, the absorbance of each sample was measuredat a wavelength of 450 nm using a microplate reader (RT-2100C,China). The 5-FU concentration producing a 50% inhibition ofproliferation (IC50) in HT-29 or SW480b6 cells for the differentexperimental formulations was calculated.

Flow cytometry apoptosis analysisCells were plated in 6-well culture plates (1 � 105 cells/well)

and incubated for 24 hours. Then, the medium was exchangedwith DMEM containing various formats of 5-FU, while cellstreated with only medium were used as control. After incubationfor 24 hours, cells were harvested, washed in PBS, and resus-pended in Annexin V binding buffer. Following the instructionsprovided by the manufacturer, Annexin V–FITC was added to thecell suspensions and the cells were incubated for 15 minutes at4�C. Then, PI was added and incubated for 5 minutes at 4�C. Thefluorescently labeled cells were tested using a flow cytometer, andthe results were analyzed by Flowjo software.

Western blottingCytochrome C release was analyzed by Western blotting. The

cytosol lysate was extracted using the cytochrome C apoptosisassay kit by the method described before (19). Cytosolic proteinsat a final protein amount of 20 mgwere loaded onto an SDS-PAGEgel and were electrophoresed. Subsequently, the separated pro-teins were transferred onto polyvinylidene difluoridemembranesand were immunoblotted using the cytochrome C primary anti-body overnight at 4�C, followed by incubation with an HRP(horseradish peroxidase)-labeled secondary antibody. The pro-tein bands were visualized using the ECL method.

The levels of caspase-3/9, cleaved caspase-3, cleaved PARP, andphosphorylated ERK1/2 (p-ERK1/2) were also determined usingWestern blotting following the same procedures that weredescribed above.

Caspase activity assayA caspase activity assay was carried out using a caspase fluo-

rometric assay kit following the instructions provided by themanufacturer. In brief, cells were homogenized on ice with lysisbuffer. An aliquot of 50 mL of supernatants was incubated with anequal volume of the reaction buffer containing fluorogenic pep-tide substrate at 37�C for 1 to 2 hours. Enzymatic release of freefluorogenic moiety was measured by a fluorometer.

Tissue distribution studyBALB/C female nude mice bearing HT-29 of which the tumor

size was larger than 100 mm3 were randomly assigned to threegroups and injected intravenously through tail veinswith adosageof 5-FU (20mg/kg), liposomesþ5-FU(20mg5-FU/kg), or ITGB6-targeted immunoliposomesþ5-FU (20 mg 5-FU/kg). Blood sam-ples were obtained from the retro-orbital plexus at predeterminedtime points, and the plasma was collected and stored. The micewere sacrificed at the indicated time points (0.5, 2, 4, 8, 12, and 24hours after the drug was administered i.v.) to collect tissuesamples. The tissues (heart, liver, spleen, lung, kidney, and tumor)were removed, washed, weighed, and homogenized in physio-logic saline. 5-FUwas extracted using ethyl acetate. The amount of5-FU in tissues was analyzed by the HPLC assay. The data werenormalized to the tissue weight. The main pharmacokineticparameters were calculated via the statistical moment methodusing DAS 2.0 software.

In vivo therapeutic efficacyBALB/C female nude mice were subcutaneously implanted

with HT-29 or SW480b6 human colon cancer cells at a finalconcentration of 1 � 107/200 mL. Mice with tumor volumes of

ITGB6-Targeted Drug Delivery for Colon Cancer Therapy

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approximately 50 mm3 were selected and randomly assigned tothree treatment groups. Free 5-FU, 5-FU–loaded liposomes, orITGB6-targeted immunoliposomes were intravenously injectedtwice a week at a 5-FU dosage of 20 mg/kg. Blank immunolipo-somes or physiologic saline (N.S.) were also injected as controls.The tumor size and body weight were measured every other dayduring the treatment period. The tumor volume was calculatedfrom the following formula: (W2 � L)/2. After 3 weeks, the micewere sacrificed and the tumors were dislodged andweighed. All ofthe animal studies were conducted using a protocol approved bythe Institutional Animal Care andUseCommittee at the School ofMedicine, Shandong University (Jinan, Shangdong, P.R. China).

TUNEL staining assayMice were sacrificed, and sections of tumor tissues in each

groupwere prepared. The TUNEL assaywas carried out using an InSitu Cell Death Detection Kit according to the manufacturer'sinstructions. Finally, the apoptotic index was calculated by count-ing the number of TUNEL-positive nuclei visible on 40� high-power-field objective in at least 3fields/sample and expressing theresults as percentage of the total number of cells in the samefields.Apoptotic cells were recognized by the appearance of brown ortan-stained nuclei.

Statistical analysisData were presented as the mean � SD and all measurements

were performed from at least three independent experiments. Thestatistical significance was determined by the Mann–Whitney testor Student t test. P < 0.05 was considered statistically significant.The statistical analyses were performed using theGraphPad Prismsoftware (GraphPad Software, Inc.).

ResultsCharacterization of liposomes

The morphology of liposomes and ITGB6-targeted immuno-liposomes were spherical or ellipsoidal, as we could directlyobserve by TEM (Fig. 1B). Size distribution of both liposomeswas shown in Fig. 1A, and themeanparticle size of ITGB6-targetedimmunoliposomes was 405.1� 2.74 nm, which was greater thanthat of liposomes (345.5 � 2.21 nm). These indicated that themAb conjugation may have a significant impact on immunolipo-somes size. The zeta potential of the liposomes was also inves-tigated. The zeta potential of the liposomes was �23.77 � 2.35mV.AftermAb conjugation, the zeta potential further decreased toa value of �8.18 � 1.83 mV. Furthermore, as the amount ofconjugatedmAbs increased, the immunoliposomes zeta potentialgradually decreased (data not shown), which most likely indi-cated that immunoliposomes stability was associated with theamount of mAbs conjugated. The 5-FU encapsulation efficiencies(EE%) of the liposomes and immunoliposomes were 78.43% �2.23% and 75.95% � 2.30%, respectively. Figure 1C showed thein vitro 5-FU release profiles from both liposomal formats in thePBS or serum condition. ITGB6-targeted immunoliposomes andliposomes had similar trends of 5-FU release, indicating that theITGB6mAbs conjugated onto the liposomes had no influence onthe kinetic properties. In addition, compared with release profilesin PBS, serum could significantly retard the release of 5-FU inliposomes. In addition, ITGB6-targeted immunoliposomesshowed no greatly sensitivity to different pHs on the release of5-FU (Fig. 1D).

Uptake of ITGB6-targeted immunoliposomes in ITGB6expressing colon cancer cells

Before researching the cellular association aspects of ITGB6-targeted immunoliposomes, we assessed the ITGB6 expressionlevel inHT-29 and SW480b6 colon cancer cells byflow cytometry.As shown in Fig. 1E, both HT-29 and SW480b6 cells obviouslyexpressed ITGB6, whereas HT-29 treated with b6-siRNA anduntreated SW480 had low or negative ITGB6 expression. SW480mock cells had lower ITGB6 expression, partially due to thenonspecific binding; however, thefluorescence valueswerewithinthe experimental error. ITGB6-targeted immunoliposomes andliposomes were then loaded with fluorescent coumarin-6 toinvestigate the interactions between the liposomes and the targetcells. The specific cell binding and cellular uptake of ITGB6-targeted immunoliposomes were visualized by fluorescencemicroscopy (27). The fluorescence images of HT-29 andSW480b6 cells were shown in Fig. 2A. The fluorescence intensitiesof ITGB6-targeted immunoliposomes in both HT-29 andSW480b6 cells were significantly greater than the liposomes,suggesting that ITGB6-targeted immunoliposomes had highercellular accumulation than liposomes. However, free couma-rin-6 had high cellular accumulation, partially due to its highlyhydrophobic nature; as a result, it was chosen as the positivecontrol (28). A flow cytometry analysis was also performed toquantify the cellular internalization of ITGB6-targeted immuno-liposomes. As illustrated in Fig. 2B, slight fluorescence wasdetected in HT-29 and SW480b6 cells incubated with liposome-sþcoumarin-6, whereas much higher fluorescence intensitieswere measured in both cell lines treated with ITGB6-targetedimmunoliposomesþcoumarin-6. All above results indicated thatITGB6-targeted immunoliposomes could enhance the cellularinternalization compared with liposomes, which probablyresulted in improved anticancer efficacy in colon cancer cells.

To evaluate the correlation between ITGB6-targeted immuno-liposomes cellular uptake and ITGB6 expression and to confirmthe specificity of ITGB6-targeted immunoliposomes towardITGB6-expressed colon cancer cells, HT-29 cells of which ITGB6was suppressed by siRNA or IGTB6-negative SW480 cells werealso both treated with ITGB6-targeted immunoliposome-sþcoumarin-6. Figure 2A also showed that fluorescence inITGB6-suppressed HT-29 or ITGB6-negative SW480 cells wassignificantly weaker compared with HT-29 or SW480b6 cells afterITGB6-targeted immunoliposomesþcoumarin-6 treatment. Thedifferences observed between ITGB6-targeted immunoliposomesinternalization in cells that expressed different levels of ITGB6may testify the specific cell binding of ITGB6-targeted immuno-liposomes and indicate that the cellular uptake was dependent onthe ITGB6 expression on cellular surface.

Cytotoxicity and proliferation inhibition on colon cancer cellsTo evaluate the antitumor activity of ITGB6-targeted immu-

noliposomes, colon cancer cells were exposed to 5-FU,liposomesþ5-FU, and ITGB6-targeted immunoliposomesþ5-FU, with 5-FU at an equivalent dose from 0.2 to 125 mg/mL.The cytotoxicity and growth inhibition of the cells was thenevaluated by the CCK-8 assay. As shown in Fig. 2C, alltreatments demonstrated a clear 5-FU dose-dependent cyto-toxicity in the experimental cell lines. However, for HT-29 andSW480b6 cells, the ITGB6-targeted immunoliposomesþ5-FUinhibited the cellular growth more greatly than liposomesþ5-FU (P < 0.01). Interestingly, the difference in growth

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Figure 2.A, fluorescence microscopy images showed the internalization of ITGB6-targeted immunoliposomes (IL) and liposomes (Ls) in HT-29 and SW480b6 colon cancercells, and the internalization of ITGB6-targeted immunoliposomes in HT-29 b6-siRNA or SW480 cells on which IGTB6 was low expressed or negative. Cellnuclei were stained blue with DAPI and coumarin-6 was shown as green fluorescence. (Continued on the following page.)

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inhibition between these two types of liposomes was almostundistinguishable in the ITGB6-negative SW480 cells(Fig. 2D), which implied that the improved antitumor activityof ITGB6-targeted immunoliposomes may be attributed to thespecific target binding to ITGB6 on cellular surface. The IC50

values of 5-FU at 24 and 48 hours for HT-29 and SW480b6cells were presented in Fig. 2E. The ITGB6-targeted immuno-liposomes produced a more than 90% decrease in 5-FU IC50

values relative to that of liposomes, which suggested that theITGB6-targeted immunoliposomes enhanced the cytotoxicityof 5-FU against these colon cancer cells.

Also in Fig. 2C and E, compared with free 5-FU, liposomesþ5-FU was more cytotoxic against both HT-29 and SW480b6 cells (P< 0.05) and reduced the 5-FU IC50 values over 50%. These resultswere in accordance with previous studies which demonstratedthat the in vitro cytotoxicity of liposomal 5-FUwashigher than thatof the free drug (29, 30). In addition, the blank nontargetedliposomes and ITGB6-targeted immunoliposomes had no signif-icant effects on cell growth (data not shown).

Efficacy of ITGB6-targeted immunoliposomes on the 5-FU–induced apoptosis of colon cancer cells

ITGB6 could protect HT-29 and SW480b6 colon cancer cellsfrom 5-FU–induced apoptosis (19). Therefore, to confirm the 5-FU–induced apoptosis efficacy of ITGB6-targeted immunolipo-somes, HT-29, and SW480b6 cells were treated with 5-FU,liposomesþ5-FU, and ITGB6-targeted immunoliposomesþ5-FUfor 24 hours, with final 5-FU concentrations of 10 mg/mL inSW480b6 cells and 40 mg/mL in HT-29 cells. Subsequently, thecells were analyzed using flow cytometry. As presented in Fig. 3Aand B, in the ITGB6-targeted immunoliposomes groups, theapoptotic rate of HT-29 cells was 21.91% � 1.62%; the rates inthe free 5-FU and liposome groups were 8.99% � 1.00% and14.53% � 0.99%, respectively. It was evident that the ITGB6-targeted immunoliposomes causedmore apoptosis inHT-29 cellsthan the liposomes (P < 0.01). For SW480b6 cells, the apoptoticrates in the free 5-FU, liposome, and ITGB6-targeted immunoli-posome groups were 12.77% � 1.21%, 16.61% � 1.23%, and26.18% � 2.43%, respectively. There were also significant differ-ences between the liposome and immunoliposome groups (P <0.01). The apoptotic rates of bothHT-29 and SW480b6 cells in theLsþ5-FU groups were higher than in the free 5-FU groups (P <0.05). Moreover, the blank ITGB6-targeted immunoliposomesdid not affect the apoptosis in either cell line.

Because the ITGB6-mediated chemotherapeutic resistance ofcolon cancer cells was through the ITGB6–ERK pathway (19), itwas essential to verify whether the ITGB6-targeted immunolipo-somes directly affected the pathway before we speculated thereason for the enhanced 5-FU–induced apoptosis of ITGB6-tar-geted immunoliposomes. Therefore, we examined the levels ofphosphorylated ERK1/2 (p-ERK1/2) inHT-29 and SW480b6 cellstreated with ITGB6-targeted immunoliposomes or liposomesusing the Western blot method. As shown in Fig. 3C, there wereno obvious differences in p-ERK1/2 levels between the immuno-

liposomes and liposomes, which could indicate that ITGB6-targeted immunoliposomes had no influence on the ITGB6pathway. In conclusion, we confirmed that in comparison withliposomes, ITGB6-targeted immunoliposomes promoted 5-FU–induced apoptosis probably by enhancing cellular internalizationrather than inhibiting the ITGB6 pathway.

Efficacyof ITGB6-targeted immunoliposomeson the apoptosis-associated proteins in colon cancer cells

It was evident that 5-FU–induced apoptosis in colon cancercells was mediated predominantly via themitochondrial apopto-tic pathway, which involves two crucial processes: cytochrome Crelease and caspase-3 activation (31). As we previously demon-strated, ITGB6-mediated colon cancer cells' 5-FU resistance wasassociated with this apoptotic pathway (19). Therefore, to inves-tigate whether the ITGB6-targeted immunoliposomes enhanced5-FU sensitivity in colon cancer cells via this apoptotic pathway,HT-29 and SW480b6 cells were grouped and treated as describedin the section above, and the levels of mitochondrial apoptosis–related proteins were then measured by Western blotting. Asshown in Fig. 3D–G, in HT-29 cells, cytosolic cytochrome C levelwas evidently higher in the ITGB6-targeted immunoliposomegroup than the liposome group (P < 0.01). Consequently, theITGB6-targeted immunoliposomes group had higher cleavedcaspase-3 and cleaved PARP levels compared with the liposomegroup (P < 0.01), indicating the upregulation of the mitochon-drial apoptotic pathway. However, there were no significantdifferences between the liposome and free 5-FU groups. Similarresults were obtained in SW480b6 cells: cytosolic cytochrome C,cleaved caspase-3, and cleaved PARP remained at higher level inthe ITGB6-targeted immunoliposome group than the liposomegroup (P < 0.05). Unlike HT-29 cells, the liposome groups inSW480b6 cells had higher cytochrome C, cleaved caspase-3, andcleavedPARP than the free 5-FU groups (P<0.05),most likely dueto different cell characteristics. However, there were no significantdifferences in the expression levels of capase-9 and caspase-3between free 5-FU and liposomal 5-FU groups in both cell lines.In addition, the activities of caspase-9 and caspase-3 were alsodetected. In both HT-29 and SW480b6 cells, the caspase-9 andcaspase-3 activities were higher in ITGB6-targeted immunolipo-somes groups than liposome groups (Fig. 3H and I) (P < 0.05).

In conclusion, considering the result that therewere noobviousdifferences in cytochrome C expression and caspase-9/3 activitiesbetween the blank immunoliposomes and control groups, thepotential mechanism underlying the promoted mitochondrialapoptotic pathway may include enhanced 5-FU accumulation inthe target cells due to higher ITGB6-targeted immunoliposomesintracellular uptake.

Pharmacokinetic studies andbiodistributionof ITGB6-targetedimmunoliposomes

Themain pharmacokinetic parameters of both 5-FU liposomalformulations were evaluated in HT-29 human colon cancerxenograft models. The areas under the curve of blood 5-FU

(Continued.) Green fluorescence intensity in the immunoliposomes group was higher than in the liposomes group. There was no increasement of fluorescenceintensity by immunoliposomes in HT-29 b6-siRNA and SW480 cells. HT-29 con-siRNA and SW480 mock transfectant cells treated with immunoliposomesþcoumarin-6 served as control. Bar¼ 20mm.B, internalization of liposomeswere evaluatedby flowcytometry inHT-29 andSW480b6 cells. Growth inhibition in cellswas evaluated by the CCK-8 assay. All format of 5-FU inhibited colon cancer cells growth in a dose-dependent way; ITGB6-targeted immunoliposomes enhanced 5-FU–induced growth inhibition compared with liposomes in HT-29 and SW480b6 cells (C), whereas there was no evident difference between ITGB6-targetedimmunoliposomes and liposomes (Ls) in SW480 cells (D). � , P < 0.05 vs. 5-FU; ##, P < 0.01 versus liposomesþ5-FU. E, IC50 of 5-FU in HT-29 and SW480b6 cells at 24and 48 hours. Data represent mean � SD (n ¼ 5). �� , P < 0.01 versus 5-FU; ##, P < 0.01 versus liposomesþ5-FU.






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Figure 3.Efficacy of ITGB6-targeted immunoliposomes (IL) on the 5-FU–induced apoptosis in colon cancer cells. A, apoptosis of HT-29 and SW480b6 cells induced by free 5-FU, liposomes (Ls)þ5-FU and immunoliposomesþ5-FU. And quantification of cell apoptotic rates was shown (B). In both cells lines, no significant differenceswere observed in apoptosis between control and blank immunoliposomes groups. (Continued on the following page.)

Liang et al.





concentration versus time (AUC0–24) following intravenousadministration of ITGB6-targeted immunoliposomesþ5-FU andliposomeþ5-FU were 87.56 � 12.03 mg/L�h and 83.56 � 12.57mg/L�h, which were significantly higher than the free 5-FUAUC0–24 (18.65 � 3.20 mg/L�h; P < 0.01). Immunoliposomesand liposomes with half-lives of 5-FU 5.69� 1.33 hours and 5.89� 0.97 hours, respectively, increased approximately 18-fold com-pared with free 5-FU (0.32� 0.06 hours). These results indicatedthat ITGB6-targeted immunoliposomes which coupled with

ITGB6 mAbs remained the sustained release and prolongedhalf-life pharmacokinetic properties of the liposomes.

For effective tumor-targeted drug delivery, the ITGB6-tar-geted immunoliposomes should be more inclined to localizein the tumor compared with the nontargeted liposomes, whichwould result in high drug concentrations in tumor cells andspare normal tissues from unnecessary toxicity. The tissues 5-FU concentrations versus time were determined to estimate thebiodistribution and tumor localization of the ITGB6-targetedimmunoliposomes and liposomes (Fig. 4). The 5-FU concen-trations were high in heart, liver, and kidney tissues after free 5-FU injection, which probably could indicate 5-FU side effects.In addition, because 5-FU was mainly metabolized in the liver(32), a high liver accumulation of 5-FU may indicate acceler-ated drug depletion. On the contrary, both liposomal formula-tions decreased the 5-FU concentration in the heart, liver, andkidney, which would be expected to reduce 5-FU side effectsand clearance. In tumor tissues, ITGB6-targeted immunolipo-somes and liposomes had a higher 5-FU concentrations thanfree 5-FU (P < 0.01; Figs. 4 and 5). However, as shown in Fig. 5,the 5-FU concentration for ITGB6-targeted immunoliposomesin the tumor steadily increased and climaxed after 12 hours,whereas 5-FU concentrations for liposomes decreased as timeprogressed. In addition, the AUC0–24 values of ITGB6-targetedimmunoliposomes in tumor tissues were 39.28 � 0.80 mg/g�h,higher than that of liposomes (10.42� 0.62 mg/g�h), indicatingthat ITGB6-targeted immunoliposomes enhanced tumor-tar-geting efficiency compared with liposomes.

Therapeutic efficacy of ITGB6-targeted immunoliposomesin vivo

To evaluate the therapeutic efficacy of ITGB6-targeted immu-noliposomes, free 5-FU, liposomesþ5-FU, and immuno-liposomesþ5-FU at the dose of 5-FU 20 mg/kg was intrave-nously injected in mice bearing HT-29 and SW480b6 humancolon cancer cell xenografts. The antitumor effect, indicated bytumor growth, was shown in Fig. 6A and C. In comparison with

(Continued.) Immunoliposomes causedmore 5-FU–induced apoptosis than liposomes, while both immunoliposomes and liposomesweremore likely to promote theapoptosis than free 5-FU. Data represent mean � SD (n ¼ 3). � , P < 0.05; �� , P < 0.01 versus 5-FU; ##, P < 0.01 versus liposomesþ5-FU. C, activation of ERKwas examined byWestern blotting. There was a similar level of p-ERK between ITGB6-targeted immunoliposomes and Ls in HT-29 and SW480b6 cells. D, Westernblotting showed the levels of cytosolic cytochrome C, caspase-9, caspase-3, cleaved caspase-3, and cleaved PARP, in HT-29 and SW480b6 cells. Quantificationof cytochrome C (E), cleaved caspase-3 (F), and cleaved PARP (G) expression were also showed. Values were expressed as a fold of b-actin. Data representmean � SD (n ¼ 3). � , P < 0.05 versus 5-FU; #, P < 0.05; ##, P < 0.01 versus liposomesþ5-FU. H and I, caspase-9/3 activity were examined using the fluorometricprotease assay. Data represent mean � SD (n ¼ 3). � , P < 0.05 versus 5-FU; #, P < 0.05; ##, P < 0.01 versus liposomesþ5-FU.

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Liang et al.




free 5-FU, both liposomal 5-FU formulations obviously sup-pressed tumor growth during the treatment period (P < 0.05in HT-29 models and P < 0.01 in SW480b6 models). No antitu-mor effects were observed in the N.S. and blank immunolipo-some groups. Moreover, the tumor suppression of ITGB6-tar-geted immunoliposomesþ5-FU was significantly stronger thanliposomesþ5-FU (P < 0.01). Consistent with the above result,both liposomal 5-FU formulations greatly reduced thefinal tumorweight in comparison with 5-FU and the average tumor weightin mice treated with ITGB6-targeted immunoliposomesþ5-FUwas approximately 3-fold lower than that in mice treated withLsþ5-FU (P < 0.01; Fig. 6B and D). In addition, the TUNEL assaywas done to correlate the in vivo antitumor activity of ITGB6-targeted immunoliposomes with its apoptosis-inducing activity.As shown in Fig. 6E and F, compared with liposomesþ5-FU,ITGB6-targeted immunoliposomesþ5-FU–induced apoptosis intumor cells by a factor of 1.5- to 1.7-fold (P < 0.05).

The changes in mice body weights were monitored to evaluatethe side effects of the different treatments. As shown in Fig. 6G, anobvious loss of body weight was observed in mice treated with 5-FU compared with mice treated with 5-FU–loaded liposomes orimmunoliposomes (P<0.01). Bodyweight gainof liposomes andimmunoliposomes groups that was similar within the margin oferror, was not as robust as that of control, partially due to the 5-FUrelease in blood and slight accumulation of 5-FU in some othertissue. Figure 6G also demonstrated that body weight changes inthese three types of 5-FU treatment groups were inconspicuousduring the first 8 days of treatment, but differences were observedafter that time point and subsequently becoming larger as timepassed by. In addition, themice treated with free 5-FU had poorerbody conditions after the 9th or 12th day during the treatmentperiod.

DiscussionThe development of multidrug resistance (MDR) in cancer

cells and the systemic toxic side effects are major obstacles tosuccessful chemotherapy for colon cancer treatment. One of theapproaches for simultaneously overcoming MDR and reducingsystemic toxicity is to incorporate therapeutic drugs into nano-carriers, such as liposomes (33), for drug delivery into tumorsites. Liposomes carrying PEG phospholipid derivatives ontheir surfaces have prolonged blood circulation times andfacilitate the tumor tissue accumulation via the enhancedpermeability and retention effect (34, 35). However, becausethere is concern that liposomes accumulation in the tumor areamay not guarantee the bioavailability of the encapsulated drugin cancer cells or MDR cancer cells, immunoliposomes havebeen used to increase the efficiency and specificity of intracel-lular drug uptake by targeted delivery and ultimately toimprove chemotherapy efficacy (36).

ITGB6 has the necessary characteristics to be a target forliposomal drug delivery, that is, abundant expression in solidtumors and limited expression in normal epithelial tissues (17).Furthermore, ITGB6 has specific tumor-related properties that

make it a potential targeting receptor for tumor chemotherapy,particularly for colon cancer. We previously verified that ITGB6was associated with chemotherapeutic resistance in colon cancerand that ITGB6 overexpression protected colon cancer cells from5-FU–induced growth inhibition and apoptosis (19). Therefore,we used ITGB6-targeted immunoliposomes in this study as a drugdelivery to investigate whether ITGB6-targeted immunolipo-somes can selectively and specifically deliver increased drug toITGB6-positive colon cancer cells to overcome MDR and toimprove chemotherapeutic efficacy in colon cancer.

Immunoliposomes enhance the cellular internalization vialigand–receptor interactions compared with normal liposomes(37). In this study, the specific cell binding and internalization ofITGB6-targeted immunoliposomes in colon cancer cells were alsoobserved.Wedetermined that ITGB6-targeted immunoliposomeshad significantly higher cell associations in HT-29 and SW480b6cells compared with liposomes. However, the cellular internali-zation of ITGB6-targeted immunoliposomes was prominentlysuppressed in ITGB6-negative or low-expression cells, whichtestified the specific cellular internalization of ITGB6-targetedimmunoliposomes and indicated the fact that ITGB6-targetedimmunoliposomes cellular internalization depended on theITGB6 expression level on the cell surface. According to theseresults, it could be concluded that ITGB6 targeted binding wasproved to enhance the cellular uptake of liposomes in ITGB6-positive colon cancer cells, and even speculated that a higher levelof ITGB6 could possibly result in more cellular internalization ofITGB6-targeted immunoliposomes. In addition, it is well knownthat the interaction between ligand-targeted liposomes and targetcells is mediated by endocytosis (38). Therefore, as a result of theendo/exocytic cycling procedure of ITGB6 in the colon cancer cells(39), we hypothesize that upregulated cellular internalization ofITGB6-target immunoliposomes is also associated with this pro-cedure of ITGB6, which we will validate in future studies.

As a hydrophilic drug, 5-FU is absorbed in low efficiency justmainly dependent on cell membrane transporters, while theentry of liposomal 5-FU into the cells is relatively efficient byendocytosis-mediated internalization of liposomes (40, 41).This could potentially explain the fact that liposomeþ5-FUexerted greater cellular toxicity than free 5-FU (Figs. 2Cand 3A). The ITGB6-targeted immunoliposomes, which hadincreased cellular internalization compared with liposomes,could consequently enhance 5-FU accumulation in colon can-cer cells. Then, the increased 5-FU uptake would result in highercellular toxicity against colon cancer cells, as what we deter-mined in cellular toxicity and apoptosis studies. Furthermore,because of the fact that ITGB6-targeted immunoliposomescellular internalization was associated with the ITGB6 expres-sion level on cellular surface, the higher ITGB6-expressed coloncancer cells, which were more likely to produce 5-FU resistance,would probably have a higher level of 5-FU cellular accumu-lation. In addition, it has previously been reported thatthe suppression of the mitochondrial apoptotic pathway wasthe preferred mechanism of chemotherapeutic resistance for themajority of anticancer drugs, including 5-FU (42, 43). Because

(Continued.) Both liposomesþ5-FU and immunoliposomesþ5-FU were significantly superior to free 5-FU and tumor suppression of immunoliposomesþ5-FU wasgreater than liposomesþ5-FU. B and D, tumors were dislodged and weighed. Immunoliposomesþ5-FU groups had a lower tumor weight than liposomesþ5-FUgroups. Data represent mean � SD (n ¼ 5). � , P < 0.05; �� , P < 0.01 versus 5-FU; ##, P < 0.01 versus liposomesþ5-FU. HT-29 and SW480b6 xenografts weredone with TUNEL staining (E), and quantification of percentage of apoptotic cells was calculated (F). � , P < 0.05 versus 5-FU; #, P < 0.05 versus liposomesþ5-FU. G,body weight changes during the treatment. Free 5-FU had more serious effects on body weight than liposomesþ5-FU or immunoliposomesþ5-FU, whilethere was no difference between liposomesþ5-FU and immunoliposomesþ5-FU. Data represent mean � SD (n ¼ 5). �� , P < 0.01 versus 5-FU.






we demonstrated that the expression levels of the mitochon-drial apoptotic-related proteins (cytochrome C, cleaved cas-pase-3, and cleaved PARP) and activities of caspase-9/3 wereincreased after ITGB6-targeted immunoliposomesþ5-FU treat-ment, we speculated that ITGB6-targeted immunoliposomesmay overcome drug resistance by facilitating mitochondrialapoptotic pathway activation.

These promising in vitro results were further evaluated in vivo.The antitumor efficacy of ITGB6-targeted immunoliposomes wassignificantly superior to that of liposomes, which was probablycaused by increased drug accumulation in tumor issues. Althoughwe determined that both types of liposomal 5-FU had similarpharmacokinetic properties in vivo, the 5-FU concentration in thetumor tissues of mice treated with ITGB6-targeted immunolipo-somes was consistently higher than liposomes. These increased 5-FU level observed in the tumor tissues was probably achieved bythe following potential mechanism. Both liposomes are able toaccumulate in tumor tissues, ultimately reaching high levels in thetumor site. The nontargeted liposomes passively interact with thetumor cells, eventually resulting in the delivery of the majority ofdrug into some normal tissues or cells rather than the tumor (44).Conversely, ITGB6-targeted immunoliposomes are internalizedmore efficiently due to target binding and ITGB6 endocytosis,which greatly increase the accumulation of the loaded drug incancer cells. The body weight was additionally observed to eval-uate the side effects. Although body weight changes in immuno-liposomes and liposomes groups were unobvious, which wasmost likely due to their similar tissue biodistributions and phar-macokinetic properties in vivo, ITGB6-targeted immunolipo-somes may be more inclined to reduce the side effects in thelong run, as a result of the target drug accumulation in cancer cells.

In conclusion, this study described a targeted drug deliverysystem for highly efficient and selective delivery of anticancerdrugs in colon cancer overexpressing ITGB6. The ITGB6-targetedimmunoliposomes achieved favorable antitumor efficacy and

insignificant systemic toxicity, followed by specific binding andinternalization in colon cancer cells. As a result, this approachmaybe useful for the delivery of various drugs for enhanced thera-peutic index against colon cancer. Moreover, because ITGB6 isalso specifically expressed in a variety of epithelial carcinomas(45–48), ITGB6-targeted immunoliposomes may represent apotential strategy for the clinical treatment of additional cancers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: B. Liang, M. ShahbazDevelopment ofmethodology: B. Liang,M. Shahbaz, Y.Wang, Z.Niu, B.Wang,Q. Sun, W. Niu, E. Liu, J. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Shahbaz, H. Gao, R. Fang, Z. Niu, Q. SunAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Shahbaz, B. Wang, E. LiuWriting, review, and/or revision of the manuscript: B. Liang, S. Liu,M. Shahbaz, H. Gao, J. WangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B. Liang, M. Shahbaz, Z. Niu, B. Wang, Q. Sun,J. NiuStudy supervision: M. Shahbaz, Z. Niu

Grant SupportThis study was supported by research grants from the National Natural

Sciences Foundation of China (No. 81272653) and the Independent Innova-tion Foundation of Shandong University (2012TS133).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 10, 2014; revised December 17, 2014; accepted December 19,2014; published OnlineFirst December 30, 2014.

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2015;21:1183-1195. Published OnlineFirst December 30, 2014.Clin Cancer Res Benjia Liang, Muhammad Shahbaz, Yang Wang, et al. CarcinomaDrug Delivery and Enhance Therapeutic Efficacy in Colon

6-Targeted Immunoliposomes Mediate Tumor-SpecificβIntegrin

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