research targeted gene silencing using rgd-labeled ... · arg-gly-asp (rgd) peptide-labeled...

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Published OnlineFirst June 10, 2010; DOI: 10.1158/1078-0432.CCR-10-0005

Cancer Therapy: Preclinical Clinical
Cancer
Research

Targeted Gene Silencing Using RGD-LabeledChitosan Nanoparticles

Hee Dong Han1, Lingegowda S. Mangala1, Jeong Won Lee1,7, Mian M.K. Shahzad1,6, Hye Sun Kim1,8,Deyu Shen1, Eun Ji Nam1,9, Edna M. Mora2,11,12, Rebecca L. Stone1, Chunhua Lu1, Sun Joo Lee1,10,Ju Won Roh1,13, Alpa M. Nick1, Gabriel Lopez-Berestein3,4,5, and Anil K. Sood1,4,5

Abstract

Authors' AOncology,5Center forTexas M.Dand Gyne7DepartmeSungkyunPathology,KwandongDepartmenMedicine,UniversityKorea; 11DPuerto RicCenter, SaGynecolog

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G. Lopez-B

CorresponOncology aof Texas M713-745-52

doi: 10.115

©2010 Am

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Do

Purpose: This study aimed to develop an Arg-Gly-Asp (RGD) peptide-labeled chitosan nanoparticle(RGD-CH-NP) as a novel tumor targeted delivery system for short interfering RNA (siRNA).Experimental Design: RGD peptide conjugated with chitosan by thiolation reaction was confirmed

by proton-NMR (H-NMR). Binding of RGD-CH-NP with ανβ3 integrin was examined by flow cytometryand fluorescence microscopy. Antitumor efficacy was examined in orthotopic mouse models of ovariancarcinoma.Results: We show that RGD-CH-NP loaded with siRNA significantly increased selective intratumoral

delivery in orthotopic animal models of ovarian cancer. In addition, we show targeted silencing of mul-tiple growth-promoting genes (POSTN, FAK, and PLXDC1) along with therapeutic efficacy in the SKO-V3ip1, HeyA8, and A2780 models using siRNA incorporated into RGD-CH-NP (siRNA/RGD-CH-NP).Furthermore, we show in vivo tumor vascular targeting using RGD-CH-NP by delivering PLXDC1-targetedsiRNA into the ανβ3 integrin–positive tumor endothelial cells in the A2780 tumor-bearing mice. Thisapproach resulted in significant inhibition of tumor growth compared with controls.Conclusions: This study shows that RGD-CH-NP is a novel and highly selective delivery system for

siRNAwith the potential for broad applications in humandisease.Clin Cancer Res; 16(15); 3910–22. ©2010AACR.

RNA interference (RNAi)-based approaches hold greatpotential for cancer therapy (1–3). Short interfering RNA(siRNA)-based therapy may allow development of a broadarmamentarium of targeted drugs against genes that aredifficult to target with other traditional approaches suchas small molecules or monoclonal antibodies. However,

ffiliations: Departments of 1Gynecologic Oncology, 2Surgical3Experimental Therapeutics, and 4Cancer Biology, andRNA Interference and Non-coding RNA, The University of. Anderson Cancer Center, and 6Department of Obstetricscology, Baylor College of Medicine, Houston, Texas;nt of Obstetrics and Gynecology, Samsung Medical Center,kwan University School of Medicine, 8Department ofCheil General Hospital and Women's Healthcare Center,University College of Medicine, 9Women's Cancer Clinic,t of Obstetrics and Gynecology, Yonsei University College ofand 10Department of Obstetrics and Gynecology, KonkukHospital, Konkuk University School of Medicine, Seoul,epartment of Surgery, School of Medicine, University ofo, and 12University of Puerto Rico Comprehensive Cancern Juan, Puerto Rico; and 13Department of Obstetrics &y, Dongguk University IIsan Hospital, Goyang, South Korea

lementary data for this article are available at Clinical Cancernline (http://clincancerres.aacrjournals.org/).

ernstein and A.K. Sood are joint senior authors.

ding Author: Anil K. Sood, Departments of Gynecologicnd Cancer Biology, Unit 1362, P.O. Box 301439, University.D. Anderson Cancer Center, Houston, TX 77230-1439. Phone:66; Fax: 713-792-3643; E-mail: [email protected].

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one of the key challenges to the use of siRNA for therapyis the need for efficient intracellular delivery because un-protected siRNA is rapidly cleared or degraded by nu-cleases. Delivery of siRNA across plasma membranesin vivo has been achieved using delivery systems such asliposomes (4–6), nanoparticles (7–9), and chemicallymodified siRNA (1). Although these delivery approacheshave been shown to be effective in preclinical models,many cannot be used in clinical settings due to nonspecificdelivery, which may lead to unwanted or unexpected sideeffects. Therefore, to overcome these limitations, novel de-livery systems are needed. A desirable delivery systemshould lead to enhanced concentrations of therapeuticpayloads at disease sites, minimize concerns about off-target effects (3), and ultimately raise the therapeutic in-dex. Chitosan (CH) is particularly attractive for clinicaland biological applications due to its low immunogenici-ty, low toxicity, and biocompatibility (10, 11). In additionto its advantages such as a protonated amine group, chit-osan can increase binding efficiency with cells because ofelectrostatic interactions (12).For a targeted delivery system (3, 8, 13), various recep-

tors on the tumor cell surface have been established as atarget binding site to achieve selective delivery. One suchprotein receptor of interest is the ανβ3 integrin, which hasbeen considered for selective delivery (14–17). The ανβ3integrin is overexpressed in a wide range of tumors, and islargely absent in normal tissues, which is a desirable

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Translational Relevance

We have developed a novel method for linking theArg-Gly-Asp (RGD) peptide to chitosan nanoparticles(RGD-CH-NP) to increase selective delivery of shortinterfering RNA (siRNA). In addition, we show tar-geted silencing of multiple growth-promoting genesalong with therapeutic efficacy in orthotopic animalmodels of ovarian carcinoma. RGD-CH-NP is a noveland highly selective delivery system for siRNA with thepotential for broad applications in human disease.

Targeted Gene Silencing


feature for selective delivery. Here, we developed a cyclicArg-Gly-Asp (RGD) peptide-labeled chitosan nanoparticle(RGD-CH-NP) for tumor targeted delivery of siRNA. Thecyclic RGD has one or two ring structures, and providesconformation stability and improved binding selectivityfor the ανβ3 integrin. Moreover, cyclic peptides are lesssusceptible to biodegradation than linear RGD peptides(18, 19). In the current study, we show highly selectivedelivery of targeted nanoparticles to ανβ3 integrin–expressing cells and the therapeutic efficacy of this ap-proach in multiple ovarian cancer models.

Materials and Methods

Conjugation of RGD and chitosanConjugation of RGD (c[RGDfK (Ac-SCH2CO)], MW

719.82 Da) and chitosan (MW 50-190 KDa) is shown inFig. 1A. The RGD and chitosan were conjugated by thiola-tion reaction using the cross-linking reagentN-succinimidyl3-(2-pyridyldithio)-propionate (SPDP). Briefly, 10.5 mL of2 mg/mL chitosan solution (1% acetate buffer) were addedto 700 μg of SPDP to react theNH2 group of the chitosan for4 hours at room temperature. After that, 500 μg of RGDwere added to SPDP-activated chitosan solution for24 hours at room temperature. After this reaction, dialysiswas done for 48 hours to isolate conjugates. The conjugateswere confirmed by proton-NMR (H-NMR) (CH and CH-RGD: 1% acetic acid included D2O, RGD: DMSOd6, 500MHz, HRMAS-FT-NMR, Bruker, Germany). In addition, todetermine the RGD concentration in RGD-CH-NPs, RGDpeptide was labeled with FITC as shown in SupplementaryFig. S1 (20).

Preparation of siRNA/RGD-CH-NPRGD-CH-NP was prepared based on ionic gelation of

anionic tripolyphosphate and siRNA. Briefly, predeterminedtripolyphosphate (0.25% w/v) and siRNA (1 μg/μL) wereadded in RGD-CH solution, and the siRNA/RGD-CH-NPwere spontaneously formed under constant stirring atroom temperature. After incubation at 4°C for 40 minutes,siRNA/RGD-CH-NP was collected by centrifugation(Thermo Biofuge, Germany) at 13,000 rpm for 40 minutesat 4°C. The pellet was washed by sterile water three times

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to isolate siRNA/RGD-CH-NP, which was stored at 4°Cuntil used.

Characteristics of siRNA/RGD-CH-NPThe RGD concentration in the RGD-CH-NPs was calcu-

lated by measuring FITC intensity based on a calibrationcurve of standard concentration of FITC-labeled with RGDby a fluorescence spectrophotometer (20). The size and ζpotential of RGD-CH-NP were measured by light scatter-ing with a particle size analyzer and Zeta Plus (Brookha-ven Instrument Co.), respectively. Coincorporation ofFITC-labeled RGD and Alexa555 siRNA into siRNA/RGD-CH-NP was observed by fluorescence microscopy, and thephysical morphology of siRNA/RGD-CH-NP was observedby scanning electron microscopy.

Cell lines and siRNAThe derivation and source of the human epithelial ovar-

ian cancer cell lines SKOV3ip1, HeyA8, A2780, andA2780ip2, and murine ovarian endothelial cells (MOEC)have been previously described (4, 5, 21). The POSTNsiRNA (target sequence: 5′-GGAUCUUGUGGCC-CAAUUA-3′), FAK siRNA (target sequence: 5′-CCAC-CUGGGCCAGUAUUAU-3′), PLXDC1 siRNA (targetsequence: 5′GACACCUGCGUCCUCGA-3′), and controlsiRNA (target sequence: 5′-UUCUCCGAACGUGUCAC-GU-3′) were purchased from Sigma (22).

Binding of RGD-CH-NPsTo confirm the in vitro binding efficiency of RGD-CH-NP

against ανβ3 integrin on the cell surface, we conductedboth flow cytometry analysis and fluorescence microscopy.Tomeasure the binding efficiency of Alexa555 siRNA/RGD-CH-NP, cells were incubated for 20minutes at 4°C after na-noparticles were added, and then cells were collected bycentrifugation (1,500 rpm, 3 minutes). The binding effi-ciency was measured by flow cytometry (23, 24). To ob-serve cell binding of RGD-CH-NP, cells were fixed in achamber slide using 4% paraformaldehyde and then thecells were stained with Hoechst 33258 for 10 minutes at4°C (to stain nuclei blue) and observed under fluorescencemicroscopy (magnification, ×200; refs. 23, 24). In addition,we confirmed intracellular delivery of chitosan nanoparti-cles (CH-NP) or RGD-CH-NP by confocal microscopy.Briefly, we added CH-NP or RGD-CH-NP in cells and thenincubated for 20 minutes at room temperature. The cellswere then fixed using 4% paraformaldehyde, after whichthe cells were stained with sytox green (to stain nucleigreen) for 10 minutes at room temperature and observedunder confocal microscopy. To confirm binding of RGD-CH-NP by tumor cells, morphology of the cells was ob-served by transmission electron microscopy (23, 24).

In vivo delivery of siRNA/RGD-CH-NPDetection of uptake of Alexa555 siRNA/RGD-CH-NP

was done as described previously (25, 26). Relevant tis-sues were harvested after single injection of either controlsiRNA/CH-NP, Alexa555 siRNA/CH-NP, or Alexa555

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Fig. 1. A, conjugation of RGD to chitosan (CH). Physical properties of siRNA/RGD-CH-NPs. B, top, RGD concentration in siRNA/RGD-CH-NPs wascalculated by measuring FITC intensity based on a calibration curve of standard concentration of FITC-labeled with RGD by fluorescence spectrophotometry.Middle and bottom, size and ζ potential of siRNA/RGD-CH-NPs were measured by light scattering with a particle size analyzer and Zeta Plus, respectively.C, incorporation of FITC-labeled RGD (green) and Alexa555 siRNA (red) into siRNA/RGD-CH-NPs was observed by fluorescence microscopy(magnification, ×400, top; scale bar, 1 μm). Morphology of siRNA/RGD-CH-NP 5 was examined by scanning electron microscopy (bottom). Error bars, SE;*, P < 0.05.

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siRNA/RGD-CH-NP into SKOV3ip1-bearing mice. Uptakeefficiency was determined by the percentage of Alexa555siRNA-labeled nanoparticles location into tissue in fiverandom fields at ×200 magnification for each tumorand organ. In addition, to confirm ανβ3 integrin–medi-ated delivery of RGD-CH-NP, we carried out ανβ3 integ-rin staining in tumor tissues as described above.

Western blot analysisThe preparation of cultured cell lysates and tumor tissue

lysates has been previously described (5, 27). Protein con-centrations were determined using a BCA Protein AssayReagent Kit (Pierce Biotech.), and aliquots of 20 μg pro-tein were subjected to gel electrophoresis on 7.5% or10% SDS-PAGE gels. Transfer to membranes and immu-noblotting were carried out as described previously (28).

Orthotopic in vivo model of ovarian cancer andtissue processingFemale athymic nude mice (NCr-nu) were purchased

from the National Cancer Institute-Frederick Cancer Re-search and Development Center and maintained as previ-ously described (29). All mouse studies were approved bythe M.D. Anderson Cancer Center Institutional AnimalCare and Use Committee. The mice used for in vivo experi-ments were 8 to 12 weeks old. To produce tumors, SKO-V3ip1, HeyA8, and A2780 cells (1 × 106 cells per 0.2 mLHBSS) were injected into the peritoneal cavity (i.p.) ofmice. Mice (n = 10 per group) were monitored daily foradverse effects of therapy and were sacrificed when anyof the mice seemed moribund.To assess tumor growth, treatment began 1 week after

i.p. injection of tumor cells into the mice. Each siRNA-incorporated CH-NP or RGD-CH-NP was given twiceweekly at a dose of 150 μg/kg body weight through i.v in-jection. Docetaxel was diluted in PBS and injected i.p oncea week, at a dose of 100 μg in 200 μL. Treatment continueduntil mice became moribund (typically 4 to 5 weeks de-pending on tumor-cell). Mouse weight, tumor weight,number of nodules, and distribution of tumors in the micewere recorded at the time of sacrifice. The individuals whodid the necropsies, tumor collections, and tissue processingwere blinded to the treatment group assignments. Tissuespecimens were fixed either with formalin or optimum cut-ting temperature (Miles, Inc.) or were snap frozen.

Real time quantitative reverse transcriptase-PCRRelative expression of POSTN and FAK mRNA in mice

after treatment was determined by real-time quantitativereverse transcriptase-PCR (qRT-PCR) using 50 ng totalRNA isolated from treated tumor tissue using the RNeasyMini Kit (Qiagen). Relative expression values were ob-tained using the 2−ΔΔCT method, and normalized to con-trol for percent fold changes (30).

Immunohistochemical stainingImmunohistochemical analysis was done on tumor tis-

sue from mice that were treated by i.v. injection of siRNA/



CH-NP or siRNA/RGD-CH-NP. Procedures for immuno-histochemical analysis of cell proliferation (Ki67), micro-vessel density (MVD; CD31), and POSTN expression(POSTN antibody) were done as described previously(27, 31). All of these analyses were recorded in five ran-dom fields for each slide at ×100 magnification. In addi-tion, terminal deoxynucleotidyl transferase-mediated nickend labeling (TUNEL) was done as described previously todetermine cell apoptosis (32). The quantification of apo-ptotic cells was calculated by the number of apoptotic cellsin five random fields at ×200 magnification. All stainingwas quantified by two investigators in a blinded fashion.

Statistical analysisDifferences in continuous variables were analyzed using

Student's t test for comparing two groups, and ANOVA wasused to comparedifferences formultiple group comparisons.For values that were not normally distributed, the Mann-Whitney rank sum test was used. The statistical package forthe Social Sciences (SPSS, Inc.) was used for all statisticalanalyses. A P <0.05 was considered statistically significant.

Results

Characteristics of siRNA incorporated RGD-CH-NPsIn this study, we selected the ανβ3 integrin as a target

receptor because it is selectively expressed in a largeproportion of ovarian cancer cells and associated tumorvasculature. Therefore, we utilized a well-characterized tar-geting peptide, RGD, which can bind specifically to theανβ3 integrin (33, 34). We first conjugated the RGD pep-tide with chitosan by thiolation reaction of SPDP (Fig. 1A)and the conjugates were confirmed by H-NMR analysis(Supplementary Fig. S2). As shown in SupplementaryFig. S2C, the peaks for the chitosan of benzene group inRGD (1) and CH2 of methylene in RGD (1.5 ppm; ref. 2)were observed at 7 to 8 ppm and 1 to 2 ppm, respectively.Additionally, conjugation of RGD with chitosan and CH-NP was measured using FITC-labeled RGD by fluorescenceintensity (Supplementary Fig. S1). Conjugation yield ofRGD with chitosan was up to 60% (data not shown),and RGD concentration on RGD-CH-NP was determinedby measuring FITC intensity based on a calibration curveof standard concentration of FITC-labeled with RGD.Based on conjugation of RGD-CH, RGD-CH-NPs were

prepared. We prepared five different siRNA incorporatedRGD-CH-NPs (siRNA/RGD-CH-NP) with varying amountsof RGD (Fig. 1B). The size and ζ potential of siRNA/RGD-CH-NPs were around 200 nm and 40 mV, respectively (Fig.1B). The histogram of RGD-CH-NP 5 is shown in Supple-mentary Fig. S3. These experiments indicate that RGD con-jugation with CH-NP does not affect the formation andphysicochemical properties of siRNA/RGD-CH-NPs. Addi-tionally, the incorporation of RGD and siRNA into RGD-CH-NP 5 was confirmed by fluorescence microscopy usingFITC-labeled RGD (green) and Alexa555-labeled siRNA(red; Fig. 1C, top). The morphology of siRNA/RGD-CH-NP 5 was determined by scanning electron microscopy.




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The particles were spherical in shape and the size wasaround 200 nm (Fig. 1C, bottom).

RGD-CH-NP enhances binding efficiency on ανβ3integrin–expressing tumor cellsWe first assessed the expression of ανβ3 integrin in

ovarian cancer cell lines by flow cytometry. AlthoughA2780ip2 cells were negative, the SKOV3ip1 cells showedpositive expression on the cell membrane against theανβ3 integrin (Fig. 2A). We next studied the binding ef-ficiency of siRNA/RGD-CH-NPs in both cell lines withdifferent concentrations of RGD (Fig. 1B). As expected,little binding was observed in A2780ip2 cells at any ofthe five different formulations tested (Fig. 2B). In theανβ3-positive SKOV3ip1 cells, however, binding in-creased in a RGD concentration-dependent manner.Among the five formulations, siRNA/RGD-CH-NP 5(1.45 μg RGD/mg chitosan) showed the highest binding(Fig. 2B). Therefore, we selected siRNA/RGD-CH-NP 5for subsequent experiments. We next confirmed bindingefficiency of Alexa555-labeled (Alexa555) siRNA/RGD-CH-NP by fluorescence microscopy against tumor cells.Alexa555 siRNA/RGD-CH-NP showed higher binding ef-ficiency (94.25% induction versus CH-NP) in the SKO-V3ip1 cells compared with non–RGD-labeled CH-NP.In contrast, similar binding efficiency was observed inthe A2780ip2 cells between RGD-CH-NP and CH-NP(Fig. 2C). To observe binding of RGD-CH-NP in tumorcells, we utilized transmission electron microscopy. Inthe SKOV3ip1 cells, RGD-CH-NP showed higher bindingcompared with the ανβ3-negative A2780ip2 cells (Fig.2D). In addition, we observed intracellular delivery ofCH-NP or RGD-CH-NP using confocal microscopy.Alexa555 siRNA/RGD-CH-NP resulted in higher intracel-lular efficiency in the SKOV3ip1 cells compared withnon–RGD-labeled CH-NP (Supplementary Fig. S4).

RGD-CH-NP enhances targeted delivery totumor tissuesPrior to conducting proof-of-concept in vivo efficacy

studies, we tested the extent of in vivo delivery followinga single i.v. injection of Alexa555 siRNA/RGD-CH-NP in-to SKOV3ip1-bearing mice after 48 hours. The siRNA wasobserved in >80% of fields examined, and showed up to3-fold higher localization into tumor tissues comparedwith CH-NP (Fig. 3A). Additionally, we stained harvestedtumors for the ανβ3 integrin to evaluate colocalization.The Alexa555 siRNA/RGD-CH-NP (red) consistentlyshowed colocalization (yellow) with the ανβ3 integrin(green) in tumor tissues (Fig. 3B). In contrast, deliverywith siRNA/CH-NP showed Alexa555-positive siRNA inboth ανβ3-positive and -negative cells. These findings in-dicate that siRNA/RGD-CH-NPs indeed result in selectivedelivery into ανβ3-positive cells. We also examined otherorgans, including liver, kidney, spleen, lung, heart, andbrain, for delivery of siRNA using either CH-NP orRGD-CH-NP. However, minimal siRNA RGD-CH-NPwas observed in these organs as compared with CH-NP

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due to the greater binding specificity for RGD-CH-NP intumor tissues (Fig. 3C).

Therapeutic efficacy of gene silencing with targetedRGD-CH-NPTo determine the effectiveness of gene silencing and

potential therapeutic efficacy, we focused on targetingperiostin (POSTN), which plays a significant role in cellinvasion, survival, and angiogenesis, leading to increasedmetastasis of cancer cells (35). The SKOV3ip1 and A2780models were selected for these experiments as they haveincreased POSTN levels (Supplementary Fig. S5). Follow-ing a single i.v. injection of POSTN siRNA/RGD-CH-NP(150 μg siRNA/kg body weight) into SKOV3ip1-bearingmice, tumors were harvested. POSTN expression was re-duced by >51% in RGD-CH-NP–treated tumors comparedwith control siRNA/CH-NP and by >20% compared withCH-NP at 24 hours (Fig. 4A). On the basis of this result,we evaluated POSTN expression by immunohistochemistryanalysis. Delivery of POSTN siRNA with RGD-CH-NPresulted in significantly greater inhibition of POSTN expres-sion in tumor tissues as compared with POSTN siRNA/CH-NP or control siRNA/CH-NP at 24 hours (Fig. 4B).We next examined the therapeutic efficacy of POSTN si-

lencing with POSTN siRNA/RGD-CH-NP in mice bearingorthotopic SKOV3ip1 (ανβ3-positive) or A2780 (ανβ3-negative) tumors. Seven days following injection of tumorcells into the peritoneal cavity, the mice were randomly al-located to the following groups (n = 10 mice/group): (a)control siRNA/CH-NP + PBS; (b) POSTN siRNA/CH-NP +PBS; (c) POSTN siRNA/RGD-CH-NP + PBS; (d) controlsiRNA/CH-NP + docetaxel; (e) POSTN siRNA/CH-NP +docetaxel; and (f) POSTN siRNA/RGD-CH-NP + docetaxel.All mice were sacrificed when animals in any group ap-peared moribund (4 to 5 weeks after cell injection depend-ing on the cell line). In the SKOV3ip1 model, POSTNsiRNA/RGD-CH-NP + PBS resulted in significant inhibi-tion of tumor growth compared with POSTN siRNA/CH-NP + PBS (24% reduction, P < 0.04) and control siRNA/CH-NP + PBS (71% reduction, P < 0.001). Notably, com-bination of POSTN siRNA/RGD-CH-NP + docetaxelshowed the greatest inhibition of tumor growth comparedwith control siRNA/CH-NP + docetaxel (32% reduction,P < 0.006) and CH-NP + docetaxel (22% reduction, P <0.01; Fig. 5A). After treatment, the decrease in POSTNmRNA level was confirmed by qRT-PCR (Fig. 5A). In theA2780 model, POSTN siRNA/RGD-CH-NP + PBS showedsignificant inhibition of tumor growth compared withcontrol siRNA/CH-NP + PBS (73% reduction, P < 0.01),however, POSTN siRNA/RGD-CH-NP + PBS showed noadditional benefit compared with POSTN siRNA/CH-NP +PBS (P < 0.22; Fig. 5B). As above, decrease in POSTNmRNA level was confirmed by qRT-PCR (Fig. 5B). Therewere no differences in total body weight, feeding habits,or behavior between the groups, suggesting that there wereno overt toxicities related to therapy.To determine potential mechanisms underlying the effi-

cacy of siRNA/RGD-CH-NP therapy in tumor tissues, we

Clinical Cancer Research





Fig. 2. Binding of siRNA/RGD-CH-NPs against ovarian cancer cells in vitro. A, ανβ3 integrin expression on SKOV3ip1 or A2780ip2 cells by flow cytometry.Cells were incubated in RPMI-1640 supplemented with 10% fetal bovine serum at 37°C for 24 hours, and then washed and incubated with 2.5 μg ofeither Alexa555-labeled siRNA/CH-NPs or siRNA/RGD-CH-NPs in PBS for 20minutes at 4°C. B, binding of siRNA/RGD-CH-NPswith different concentrationsof RGDpeptide by flow cytometry. C, binding of Alexa555 siRNA/RGD-CH-NPs andAlexa555 siRNA/CH-NP in SKOV3ip1 or A2780ip2 cells. Cells were fixed ina chamber slide using 4%paraformaldehyde and then nuclei (blue) were stainedwithHoechst 33258 for 10minutes, and bindingwas analyzed by fluorescencemicroscopy (magnification, ×200). Quantitative differences were analyzed by fluorescence intensity of Alexa555 (red)/Hoechst 33358 (blue). Error bars, SE;*, P < 0.01. D, binding of RGD-CH-NPs in SKOV3ip1 or A2780ip2 cells by transmission electron microscopy (N, nucleus; arrows, nanoparticles; bar, 2 μm).

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Fig. 3. In vivo delivery of Alexa555 siRNA/RGD-CH-NP. Tumor and organ tissues were harvested after single injection of either control siRNA/CH-NP,Alexa555 siRNA/CH-NP, or Alexa555 siRNA/RGD-CH-NP into SKOV3ip1-bearing mice. Uptake efficiency was determined by the percentage ofAlexa555 siRNA-labeled nanoparticles in five random fields (original magnification, ×200). A, tumor. C, various organs. B, colocalization of Alexa 555 siRNA/RGD-CH-NP or CH-NP (red) and ανβ3 integrin (green) in tumor tissues (original magnification, ×200). All of these analyses were recorded in five randomfields for each slide. Error bars, SE; *, P < 0.05.






examined tumors for markers of cell proliferation (Ki67),MVD (CD31), and apoptosis (TUNEL). In the SKOV3ip1model, POSTN siRNA/RGD-CH-NP + PBS showed signif-icant inhibition of cell proliferation (P < 0.001 versus con-trol siRNA/CH-NP + PBS; P < 0.05 versus POSTN siRNA/CH-NP + PBS), MVD (P < 0.001 versus control siRNA/CH-NP + PBS; P < 0.01 versus POSTN siRNA/CH-NP + PBS),and increased apoptosis (P < 0.001 versus control siRNA/CH-NP + PBS; P < 0.002 versus POSTN siRNA/CH-NP +PBS). The combination group of POSTN siRNA/RGD-CH-NP + docetaxel had significantly reduced cell prolifer-ation (P < 0.05) and MVD (P < 0.001), and increasedapoptosis as compared with the single-treatment groups(P < 0.001; Fig. 5C). In the A2780 model, POSTN siRNA/RGD-CH-NP + docetaxel showed significant inhibition ofcell proliferation (P < 0.003), MVD (P < 0.001), and in-creased cell apoptosis compared with control siRNA/CH-NP + PBS (P < 0.004). As expected, neither POSTN siRNA/RGD-CH-NP nor POSTN siRNA/CH-NP showed any sig-nificant effects on cell proliferation (P < 0.10) and apopto-sis (P < 0.33; Supplementary Fig. S6A).To establish that the effects of RGD-CH-NP are not

unique to just one target, we also did in vivo experimentswith siRNA against additional targets. We targeted FAKdue to its prominent role in ovarian cancer growth and pro-gression (36). TheHeyA8 cells were both ανβ3 integrin andFAK positive (37). Mice were randomly allocated to one offollowing six groups (n = 10 mice/group): (a) controlsiRNA/CH-NP + PBS; (b) FAK siRNA/CH-NP + PBS; (c)FAK siRNA/RGD-CH-NP + PBS; (d) control siRNA/CH-NP+ docetaxel; (e) FAK siRNA/CH-NP + docetaxel; and (f) FAKsiRNA/RGD-CH-NP + docetaxel. Treatment with FAK



siRNA/RGD-CH-NP + PBS resulted in significant inhibitionof tumor growth as compared with FAK siRNA/CH-NP +PBS (P < 0.04) and control siRNA/CH-NP + PBS (P <0.001; Fig. 5D). Combination of FAK siRNA/RGD-CH-NP +docetaxel resulted in the greatest effect on tumor growthcompared with the other single-treatment groups (P <0.02) and FAK siRNA/CH-NP + PBS (P < 0.01; Fig. 5D).After treatment, FAK mRNA levels (by qRT-PCR) werefound to be significantly lower in the FAK siRNA–treatedgroups (Fig. 5D). Treatment with FAK siRNA/RGD-CH-NP+ docetaxel resulted in significant inhibition of cell prolif-eration (P < 0.05), MVD (P < 0.01), and increased cell ap-optosis (P < 0.01). The combination group of FAK siRNA/RGD-CH-NP + docetaxel showed even further decreases incell proliferation (P < 0.001) and MVD (P < 0.03), andincreased apoptosis compared with the single-treatmentgroup (P < 0.04; Supplementary Fig. S6B).

RGD-CH-NP targets tumor vasculatureBecause the ανβ3 integrin is known to be selectively

expressed in the tumor vasculature, we also selectedPLXDC1, a target we recently identified as being upregu-lated in ovarian cancer vasculature (38). We first con-firmed that the mouse ovarian endothelial cells (in vitro)and mouse origin tumor vasculature in A2780 tumor tis-sue (in vivo) express the ανβ3 integrin although the A2780cells were negative for ανβ3 integrin expression (Supple-mentary Fig. S7A and B). As expected, the A2780 tumorsharvested from mice lacked ανβ3 expression on tumorcells; however, it was clearly present in tumor vasculaturecompared with the corpus luteum in the mouse ovary(Supplementary Fig. S7B). Prior to in vivo experiments,

Fig. 4. Effect of POSTNdownregulation following i.v.injection of POSTN siRNA/RGD-CH-NP into SKOV3ip1-bearingmice. A, Western blot analysis wasdone for POSTN expression intumor tissue (20 μg of proteinused). Quantitative differenceswere determined by densitometryanalysis. B, POSTN expression intumor tissues was assessed byimmunohistochemistry at 24 hours.All of these analyses wererecorded in five random fields foreach slide and quantitativedifference was determined bypositive/negative expression ofcells for staining (magnification,×100). Error bars, SE; *, P < 0.05.




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Fig. 5. Effect of siRNA/RGD-CH-NP on ovarian cancer growth. Treatment was started 1 week after i.p. injection of tumor cells into mice. A, SKOV3ip1.B, A2780. Either siRNA-incorporated into CH-NP or RGD-CH-NP was given twice weekly at a dose of 150 μg/kg body weight through i.v. injection.Docetaxel was diluted in PBS and injected i.p. once per week, at a dose of 100 μg, in 200 μL of PBS. Treatment was continued until mice in any groupbecame moribund (typically 4 to 5 weeks depending on tumor cell). The fold change in levels of POSTNmRNA represents the mean of triplicate experimentsby qRT-PCR. C, immunohistochemistry for cell proliferation (Ki67; magnification, ×100), microvessel density (CD31; magnification, ×100), and TUNEL(bar, 50 μm) was done on SKOV3ip1-tumor tissues following treatment with POSTN siRNA/RGD-CH-NP or CH-NP. All of these analyses were recorded infive random fields for each slide. D, HeyA8 tumor model treated with FAK siRNA/RGD-CH-NP or CN-NP. The fold change in FAK mRNA levels representsthe mean of triplicate experiments by qRT-PCR. Error bars, SE; *, P < 0.05.






we confirmed delivery of Alexa555 siRNA/RGD-CH-NPinto the tumor vasculature after injection into A2780-bearing mice. Alexa555 siRNA/RGD-CH-NP showed co-localization (yellow) with endothelial cells (CD31,green) in the tumor vasculature compared with CH-NP(Supplementary Fig. S7C). We next carried out experi-ments with siRNA targeting PLXDC1 (38), which was in-corporated into RGD-CH-NP. For these experiments,A2780 tumor–bearing mice were randomly allocated toone of the following three groups (n = 10 mice/group):(a) control siRNA/CH-NP; (b) PLXDC1 siRNA/CH-NP;and (c) PLXDC1 siRNA/RGD-CH-NP. Treatment withPLXDC1 siRNA/RGD-CH-NP resulted in significant inhi-bition of tumor growth compared with control siRNA/CH-NP (87% reduction, P < 0.001; Fig. 6A). The targeted



delivery of PLXDC1 siRNA with RGD-CH-NP resulted ineven greater efficacy compared with PLXDC1 siRNA withCH-NP (P < 0.01; Fig. 6A). Because the A2780 cells lackανβ3 integrin expression, these results suggest that theRGD-mediated CH-NP targeting is highly effective in target-ing the tumor vasculature. We next examined the effects ofPLXDC1 gene silencing on the tumor vasculature. PLXDC1siRNA/RGD-CH-NP resulted in increased apoptosis in thetumor vasculature compared with PLXDC1 siRNA/CH-NP(Fig. 6B). Additionally, to confirm PLXDC1 silencing in thetumor vasculature following PLXDC1 siRNA/RGD-CH-NPinjection into A2780-bearing mice, we carried out dual im-munofluorescence staining for endothelial cells (CD31,red) and PLXDC1 (green). The PLXDC1 siRNA/RGD-CH-NP–treated group resulted in complete PLXDC1 silencing

Fig. 6. Therapeutic efficacy ofsiRNA/RGD-CH-NP againstA2780-bearing mice. A, antitumoreffect of mouse PLXDC1 siRNA/RGD-CH-NP or CH-NP in theA2780 tumor model. Error bars,SE; *, P < 0.05. B, effect ofPLXDC1 siRNA/RGD-CH-NPtreatment on apoptosis in thetumor vasculature. Tumor sectionswere stained with CD31 (red) andTUNEL (green) using doubleimmunofluorescence staining.Colocalization of endothelial cellsundergoing apoptosis appearsyellow (magnification, ×400). All ofthese analyses were recorded infive random fields for each slideand quantitative differences wereanalyzed by positive expression ofTUNEL/CD31. C, tumor sectionswere stained with CD31 (red)and PLXDC1 (green) usingimmunofluorescence staining toexamine PLXDC1 silencing in thetumor vasculature (magnification,×400).




Han et al.

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in the tumor vasculature compared with PLXDC1 siRNA/CH-NP (Fig. 6C).

Discussion

We show here that a novel receptor-targeted deliverysystem (RGD-CH-NP) loaded with siRNA targeted to keyovarian cancer–associated genes leads to potent antitumorefficacy in ovarian carcinoma. This approach has broadutility for selectively targeting tumor cells as well as theassociated endothelial cells. RGD-CH-NP was effective insilencing multiple targets of interest and in achieving ther-apeutic efficacy.RNAi-based cancer therapy is a highly specific method

of gene silencing, but hurdles related to systemic in vivodelivery of siRNA need to be overcome to realize its fullpotential in clinical settings. Moreover, delivery efficiencyof free siRNA without the use of a nanoparticle is quitelow, and most of the free siRNA is rapidly degraded fol-lowing i.v. injection (5, 39). Therefore, to overcome thislimitation, selective targeted delivery systems are needed.Although a number of nanoparticle systems have been uti-lized for therapeutic applications, most of these are widelydistributed in the body, and could lead to undesirabletoxicities in normal tissues. In addition, wide drug distri-bution may require higher doses for gene silencing in thetarget tissue of interest. Therefore, to overcome these lim-itations against conventional passive delivery, targeted de-livery is highly desirable.Targeted delivery systems have been designed to in-

crease or facilitate uptake into target tissue (3), and to pro-tect siRNA payloads and inhibit nonspecific delivery (3).Recent work comparing nontargeted and targeted nano-particles has shown that the primary role of the targetingligands is to enhance selective cellular uptake into cancercells and to minimize accumulation in normal tissues(40). The addition of targeting ligands that provide specif-ic nanoparticle-cell surface interactions can play a vital rolein the ultimate location of nanoparticles. For example, na-noparticles can be targeted to cancer cells if their surfacescontain moieties such as peptides, proteins, or antibodies.These moieties can bind with cancer cell-surface receptorproteins such as transferrin (41) or folate (42) receptors,which are known to be increased in number on a widerange of cancer cells. These targeting ligands enable nano-particles to bind to cell-surface receptors and penetratecells by receptor-mediated endocytosis. However, a limit-ed number of nanoparticle systems have reached clinicaldevelopment (40).Nanoparticles can carry a large payload of drugs as com-

pared with antibody conjugates (40, 43). Furthermore, na-noparticle payloads are frequently located within theparticles, and their type and number may not affect thepharmacokinetics and biodistribution of the nanoparti-cles. This is unlike molecular conjugates in which the typeand number of therapeutic entities conjugated to the tar-geting ligand significantly modify the overall properties ofthe conjugate.



Several synthetic materials have been proposed for effec-tive nonviral siRNA delivery systems such as lipid-basedparticles, oligofectamine, cyclodextrin, polyethyleneimine,and cholesterol (3). Although many types of compoundshave potential utility as delivery agents, some have con-cerns regarding safety. For example, toxicity of cationiclipid particles has been reported both in vitro and in vivo(44, 45), and some synthetic agents have been found to in-duce a gene signature of their own that might increase theoff-target effects of siRNA (46, 47). Therefore, developmentof siRNA therapeutics for cancer treatment requires clinic-ally suitable, safe, and effective drug delivery systems.Chitosan nanoparticles (CH-NP) have been recently

developed for siRNA delivery (12, 48, 49). Chitosan isan attractive nanoparticle for siRNA delivery because itspositive charge allows transport across cellular mem-branes and subsequent endocytosis. Moreover, chitosanis biodegradable, biocompatible, and has low immuno-genicity (10, 12, 48). Chitosan nanoparticles without atherapeutic payload have no effect on tumor growthcompared with untreated animals (50). The ανβ3 integ-rin is known to be overexpressed in most cancer cells andthe tumor vasculature (33, 37, 51). The ανβ3 integrin isa family of cell surface receptors, which plays an impor-tant role in tumor biology and may serve as a useful tar-get (13). In addition to its role in cell matrix recognition,the ανβ3 integrin has been a focus for drug delivery strat-egies because it assists with internalization and genetransfer (33). In the current study, we developed andcharacterized RGD-CH-NP incorporated with siRNA asan ανβ3 integrin–targeted delivery system because of itshigh affinity and highly specific binding. Indeed, our tar-geted delivery-mediated gene silencing significantly en-hanced antitumor therapeutic efficacy compared with anontargeted delivery system in preclinical ovarian cancermodels. Although the RGD ligand can be useful for bind-ing to integrin family members, additional targetingapproaches may be useful. Nevertheless, the targeted de-livery strategy presented here has broad potential as a de-livery platform in human disease and could be adaptedfor other targeting ligands.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Nicholas B. Jennings and Donna Reynolds for their technicalexpertise, and Drs. Robert Langley and Michael Birrer for helpfuldiscussions.

Grant Support

NIH grants (CA 110793, 109298, 128797, and RC2GM 092599),DOD (OC-073399, W81XWH-10-1-0158), the Ovarian Cancer ResearchFund, Inc. (Program Project Development Grant), U. T. M. D. AndersonCancer Center SPORE in ovarian cancer (P50CA083639), the ZarrowFoundation, the Medlin Foundation, and the Betty Anne Asche MurrayDistinguished Professorship. A.M. Nick and R. Stone are supported by






NCI-DHHS-NIH T32 Training Grant (T32 CA101642). M.M. Shahzad wassupported by the Baylor WRHR grant (HD050128) and the GCF Molly-Cade ovarian cancer research grant.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked



advertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Received 01/01/2010; revised 05/10/2010; accepted 05/18/2010;published OnlineFirst 06/10/2010.

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2010;16:3910-3922. Published OnlineFirst June 10, 2010.Clin Cancer Res Hee Dong Han, Lingegowda S. Mangala, Jeong Won Lee, et al. NanoparticlesTargeted Gene Silencing Using RGD-Labeled Chitosan

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