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Therapeutics, Targets, and Chemical Biology

"OA02" Peptide Facilitates the Precise Targeting ofPaclitaxel-Loaded Micellar Nanoparticles to Ovarian CancerIn Vivo

Kai Xiao1,7, Yuanpei Li1, Joyce S. Lee1,4, Abby M. Gonik2, Tiffany Dong1, Gabriel Fung1, Eduardo Sanchez1,Li Xing5, Holland R. Cheng5, Juntao Luo6, and Kit S. Lam1,3

AbstractMicellar nanoparticles based on linear polyethylene glycol (PEG) block dendritic cholic acids (CA) copolymers

(telodendrimers), for the targeted delivery of chemotherapeutic drugs in the treatment of cancers, are reported.Themicellar nanoparticles have been decorated with a high-affinity "OA02" peptide against a-3 integrin receptorto improve the tumor-targeting specificity which is overexpressed on the surface of ovarian cancer cells. "Clickchemistry" was used to conjugate alkyne-containing OA02 peptide to the azide group at the distal terminus of thePEG chain in a representative PEG5k-CA8 telodendrimer (micelle-forming unit). The conjugation of OA02 peptidehad negligible influence on the physicochemical properties of PEG5k-CA8 nanoparticles and as hypothesized,OA02 peptide dramatically enhanced the uptake efficiency of PEG5k-CA8 nanoparticles (NP) in SKOV-3 and ES-2ovarian cancer cells via receptor-mediated endocytosis, but not in a-3 integrin-negative K562 leukemia cells.When loadedwith paclitaxel, OA02-NPs had significantly higher in vitro cytotoxicity against both SKOV-3 and ES-2 ovarian cancer cells as comparedwith nontargeted nanoparticles. Furthermore, the in vivo biodistribution studyshowed OA02 peptide greatly facilitated tumor localization and the intracellular uptake of PEG5k-CA8 nano-particles into ovarian cancer cells as validated in SKOV3-luc tumor–bearingmice. Finally, paclitaxel (PTX)-loadedOA02-NPs exhibited superior antitumor efficacy and lower systemic toxicity profile in nudemice bearing SKOV-3tumor xenografts, when compared with equivalent doses of nontargeted PTX-NPs as well as clinical paclitaxelformulation (Taxol). Therefore, OA02-targeted telodendrimers loaded with paclitaxel have great potential as anew therapeutic approach for patients with ovarian cancer. Cancer Res; 72(8); 2100–10. �2012 AACR.

IntroductionOvarian cancer is the ninth most common cancer, with an

estimated 22,280 new cases in 2012, but is the fifthmost deadly,with an estimated 15,500 deaths in 2012 (1). The standardtreatment for patients with advanced-stage disease usually

involves surgical staging and debulking followed by adjuvantchemotherapy, typically with platinum and paclitaxel (PTX).However, the more extensive use of chemotherapeutic drugssuch as paclitaxel is often limited by its severe side effects,including hypersensitivity reactions, myelosuppression, andneurotoxicity, which may be attributed to their nonspecificsystemic organ distribution and inadequate intratumor con-centrations, resulting in suboptimal efficacy (2, 3). Despite theintensive chemotherapy, more than 70% of patients withovarian cancer will suffer from disease relapse or recurrence,and ultimately die of this disease. Therefore, there is a tre-mendous incentive to refine existing treatment modalities toavoid or delay the recurrence and to treat recurrent ovariancancer more effectively. Optimization of chemotherapeuticdrug delivery is among the critical approaches to improve thetherapeutic index of cytotoxic agents.

Nanotechnology is an emerging field that has shown greatpromise in the development of novel diagnostic and thera-peutic agents for a variety of diseases, including cancers (4). Asthe vasculature in tumors is known to be leaky, and the tumorlymphatic system is also deficient, nanoparticles can prefer-entially accumulate in the tumor site via the enhanced per-meability and retention (EPR) effects (5). Polymeric micellesrepresent one of the most promising nanocarriers due to theirunique core-shell structure formed by amphiphilic block

Authors' Affiliations: 1Department of Biochemistry and Molecular Medi-cine, 2Division of Gynecologic Oncology, Department of Obstetrics andGynecology, 3Division of Hematology and Oncology, Department of Inter-nal Medicine, 4Pharmacy, and 5Molecular and Cellular Biology, Universityof California Davis, Sacramento, California; 6Department of Pharmacology,SUNY Upstate Cancer Research Institute, SUNY Upstate Medical Univer-sity, Syracuse, New York; and 7National Chengdu Center for SafetyEvaluation of Drugs, West China Hospital, Sichuan University, Chengdu,China

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

K. Xiao and Y. Li contributed equally to this work.

Corresponding Authors: Kit S. Lam, Department of Biochemistry &Molecular Medicine, University of California at Davis, Suite 2301, 2700Stockton Blvd, Sacramento, CA 95817. Phone: 916-734-0910; Fax: 916-734-4418; E-mail: [email protected]; and Juntao Luo, Depart-ment of Pharmacology, SUNY Upstate Cancer Research Institute, SUNYUpstate Medical University, WHA 6299, 750 East Adams Street, Syracuse,NY 13210. E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-3883

�2012 American Association for Cancer Research.

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copolymers, which could facilitate the solublization of poorlysoluble drugs and protect the drugs from degradation andmetabolism. We have recently developed a series of novellinear dendritic block copolymers (telodendrimers) compris-ing polyethylene glycol (PEG) and dendritic cholic acids (CA),which can encapsulate high concentrations of hydrophobicdrugs such as paclitaxel and self-assemble to form stable core-shell micelles under aqueous condition (6–12). The represen-tative PEG5k-CA8 micellar nanoparticles possess the idealproperties for drug delivery, including high drug loadingcapacity, optimal particle size (20–60 nm), outstanding stabil-ity (more than 6 months at 4�C), and sustainable drug releaseprofile. Paclitaxel-loaded PEG5k-CA8 nanoparticles have beenshown to exhibit superior antitumor efficacy and toxicityprofile than free drug (Taxol) and paclitaxel/human serumalbumin nanoaggregate (Abraxane) at equivalent paclitaxeldoses, in nude mice bearing human ovarian cancer (SKOV-3)xenografts (6).To further facilitate the residence, penetration, and cancer

cell uptake of delivered drugs within the tumor sites for moreefficient cancer treatment, an attractive approach is to deco-rate the nanoparticles surface with targeting ligands thatspecifically recognize receptors on cancer cells (active target-ing). Active targeting might result in higher retention ofnanoparticles drugs at tumor sites (i.e., by reducing passivetransport away from tumor) and enhanced uptake of the drugsby cancer cells via receptor-mediated endocytosis (13–15).Furthermore, actively targeted nanoparticles have also shownthe potential to overcome multidrug resistance via bypassingof P-glycoprotein–mediated drug efflux (16). Combining pas-sive and active targeting in a single platform will furtherimprove the therapeutic index of nanocarrier delivered drugs(17, 18). A wide variety of targeting ligands, including anti-bodies and single-chain Fv fragment (19, 20), peptides (21, 22),small molecules (23), and aptamers (24, 25) have been usedwith varying degrees of success to functionalize nanoparticlesfor their potential application in targeted cancer therapy.Although antibodies or antibody fragments are effective astargeting agents, there are some innate problems such asdecreased receptor affinity as a result of conjugation methods,potential immunogenicity, nonspecific uptake by reticuloen-dothelial system, and relative poor stability (17). In contrast,peptides or peptidomimetics with high binding affinity andspecificity to cancer cells may have many favorable character-istics, including deep tumor penetration due to the smallersize, lack of immunogenicity, easy synthesis and scale-up, andgood stability especially if D-configuration and unnatural ami-no acids are used (26).Integrins are a family of heterodimeric transmembrane

glycoproteins involved in a wide range of cell-to-extracellularmatrix (ECM) and cell-to-cell interactions (27, 28). It has beenfound that integrins are overexpressed on various cell typessuch as angiogenic endothelial cells and certain cancer cells.For example, a-3 integrin is overexpressed in several types ofcancers, especially ovarian cancer, breast cancer, and mela-noma (29). The overexpression of a-3 integrin on these cancercells has been exploited as a promising pharmacologic targetfor the selective drug delivery in the treatment of these cancers.

In addition, during the cell locomotion and migration, integ-rins can undergo endocytosis after the activation with anchor-ing ligands, which may facilitate the intracellular delivery ofnanoparticles drugs into cancer cells, when these nanoparti-cles are decorated with integrin-targeting ligands. A high-affinity a-3 integrin-targeting peptide "OA02" has been iden-tified in our laboratory through screening one-bead one-com-pound (OBOC) combinatorial peptide libraries (30). This"OA02" peptide has been shown to bind strongly to a-3integrin–overexpressing ovarian cancer cells and specificallytarget ovarian cancer xenografts (ES-2) in nude mice whenconjugated to near-infrared fluorescence (NIRF) dyes (30).

In the present study, we hypothesize that the incorporationof "OA02" peptide ligand onto our newly developed micellarnanoparticles (NP) will facilitate the precise homing of drugpayload to a-3 integrin–overexpressing ovarian cancer cells.First, the alkyne-modified "OA02" peptide was synthesized andconjugated to the azide-functionalized PEG5k-CA8 telodendri-mer via copper-catalyzed cyloaddition ("click chemistry").Then, the binding specificity, uptake efficiency, and in vivotumor–targeting property of fluorescence-labeled OA02-NPswere evaluated in human ovarian cancer cells and xenograftmouse model, respectively. Finally, the antitumor effect ofpaclitaxel-loaded OA02-NPs against ovarian cancer was stud-ied both in vitro and in vivo.

Materials and MethodsMaterials

Diamino PEG was purchased from Rapp Polymere. Cy5.5Mono NHS ester was purchased from Amersham Biosciences.Hydrophobic fluorescence dye DiD (1,10-dioctadecyl-3,3,30,30-tetramethylindodicarbocyanine perchlorate, D-307), 40,6-dia-midino-2-phenylindole (DAPI), and LysoTracker Red werepurchased from Invitrogen. Paclitaxel was purchased from AKScientific Inc. Taxol (Mayne Pharma) was obtained from theUC Davis Cancer Center Pharmacy. Cholic acid, MTT, andfluorescein isothiocyanate (FITC) and all other chemicals werepurchased from Sigma-Aldrich.

Synthesis of OA02-conjugated PEG5k-CA8 telodendrimerBoc-NH-PEG5k-CA8 telodendrimer was first synthesized as

described previously (6). N3-PEG5k-CA8 was obtained by the

coupling of 4-azidobutyric acid NHS ester to the terminus ofPEG after deprotecting Boc group of Boc-NH-PEG5k-CA8 with50% (v/v) trifluoroacetic acid (TFA) in dichloromethane. Thetelodendrimer was then dialyzed and finally lyophilized.

Alkyne-modified OA02 peptide (cdG-HoCit-GPQc-Ebes-K-alkyne) was synthesized via solid-phase synthesis on Fmoc-Rink Amide MBHA Resins using the standard Fmoc chemistryas described previously (30). 5-Hexynoic acid was coupled ontothe e-amino group of lysine on the peptide. Alkyne-modifiedOA02 peptide was conjugated to the N3-PEG

5k-CA8 teloden-drimer via CuI-catalyzed cyloaddition (21). The conjugationwas confirmed by the amino acid analysis (AAA). The molec-ular structure and molecular weight of OA02-PEG5k-CA8 tel-odendrimer were measured by 1H-NMR (nuclear magneticresonance) and matrix-assisted laser desorption/ionization–

A Novel Targeted Nanotherapeutics against Ovarian Cancer

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time-of-flight (MALDI-TOF) mass spectrometry (MS),respectively.

FITC- or Cy5.5-labeled telodendrimers were synthesizedby coupling FITC or Cy5.5 NHS ester to the amino group ofthe proximal lysine between PEG and cholic acid after theremoval of Dde protecting group by 2% (v/v) hydrazine indimethylformamide.

Preparation and characterization of paclitaxel-loadedOA02-NPs

Paclitaxel-loaded OA02-NPs (PTX-OA02-NPs) were pre-pared using the mixture (1:1) of blank PEG5k-CA8 and OA02-PEG5k-CA8 telodendrimers via a dry-down (evaporation)meth-od as described previously (6). To determine the amount ofpaclitaxel loaded in the nanoparticles, paclitaxel-loaded nano-particles were dissolved in dimethyl sulfoxide (1:9, v/v) andmeasured by high-performance liquid chromatography(HPLC). The encapsulation efficiency (EE) was calculatedaccording to the following formula:

EE (%)¼ (mass of paclitaxel encapsulated in nanoparticles/mass of paclitaxel added) � 100%

The morphology, particle size distribution, and zeta poten-tial of PTX-OA02-NPswere characterized by cryo-transmissionelectron microscopy (cryo-TEM) and dynamic light scattering(DLS,Microtrac), respectively. The in vitro drug release kineticsfrom PTX-OA02-NPs was measured by the dialysis method.Briefly, aliquots of PTX-OA02-NPs solution were injected intodialysis cartridges with themolecular weight cutoff value of 3.5kDa. The cartridges were dialyzed against 1 L PBS and shakenat 37�C at 100 rpm with activated charcoal to create an idealsink condition. The concentration of paclitaxel remained in thedialysis cartridge at different time points were measured byHPLC.

Cell culture and animalsSKOV-3, ES-2, andK562 cellswere purchased fromAmerican

Type Culture Collection. SKOV3-luc cells were obtained fromCaliper Life Sciences. All these cancer cell lines were authen-ticated by the suppliers and passaged in the laboratory forfewer than 6months after resuscitation. Cells weremaintainedin a 37�C/5%CO2 humidified chamber inMcCoy's 5A (SKOV-3,ES-2, and SKOV3-luc) or RPMI-1640 (K562) media supplemen-ted with 10% FBS.

Female nude mice, 6 to 8 weeks age, were purchased fromHarlan Laboratories. All animal protocols were approved bythe Institutional Animal Care and Use Committee. Ovariancancer xenograft mouse model was established by subcutane-ously injecting 5 � 106 SKOV-3/SKOV3-luc cells in a 100 mL ofmixture of PBS and Matrigel (1:1, v/v) at the right flank infemale nude mice.

Confocal microscopySKOV-3 and ES-2 cells were seeded in 8-well chamber slides.

When the cells were almost confluent, cells were incubatedwith 2 mmol/L FITC fluorescent-labeled nanoparticles andOA02-NPs for 2 hours at 37�C with 5% CO2, respectively. Then,cells were washed 3 times with cold PBS, fixed with 4%paraformaldehyde for 10minutes, and the nuclei were counter-

stained by DAPI. The slides were mounted with coverslips andobserved by Olympus FV1000 confocal microscopy. In anotherset of experiment, excess amount of a-3 integrin antibody orfree OA02 peptide (200 mmol/L) were added into the medium30 minutes before the incubation of 2 mmol/L FITC-labeledOA02-NPswith cells, followed by the same procedure as earlier.

Flow cytometryTo show the overexpression of a-3 integrin, SKOV-3 and ES-

2 cells were incubated with Alex Fluor 488–conjugated a-3integrin antibody (Chemicon International, 1:500) for 30 min-utes at 4�C, followed by PBS wash twice, and then resuspendedin PBS for the flow cytometric analysis.

SKOV-3, ES-2, and K562 (a-3 integrin negative) cells wereincubated with 2 mmol/L FITC-labeled nanoparticles or OA02-NPs for 2 hours at 37�C, respectively. Then the cells werewashed with PBS 3 times and resuspended in PBS for the flowcytometric analysis. A total of 10, 000 events were collected foreach sample. For peptide inhibition experiments, free OA02peptides with the final concentration from 2 to 200 mmol/Lwere added into the medium 30minutes before the incubationof 2 mmol/L FITC-labeled nanoparticles or OA02-NPs withcells.

Intracellular tracking of OA02-NPs in live ovarian cancercells

To simultaneously track the payload and carrier of OA02-NPs, DiD dyeswere encapsulated as drug surrogates into FITC-conjugated OA02-NPs. SKOV-3 ovarian cancer cells wereseeded in the coverglass chamber slides. After reaching 80%confluence, cells were incubated with DiD/FITC dual-labeledOA02-NPs. After 1.5 hours, LysoTracker Red (50 nmol/L) wasadded in the medium and the cells were further incubated foranother 30 minutes (31). Then, the live cells were observedunder the Olympus FV1000 confocal microscopy.

MTT assayMTT assay was used to evaluate the in vitro cytotoxicity of

blank/paclitaxel-loaded nontargeted nanoparticles and OA02-NPs against ovarian cancer cells (32). Cells were treated withblank/paclitaxel-loaded nanoparticles and OA02-NPs, respec-tively. After 2 hours of treatment, cells were washed with PBS 3times, and freshmedia were replaced in the plates. At 72 hours,MTTwas added to each well and further incubated for another4 hours. The absorbance at 570 nmwith a referencewavelengthof 660 nm was detected with a microplate reader. Untreatedcells served as a control. Results were shown as the average cellviability [(ODtreat � ODblank)/(ODcontrol � ODblank)� 100%] oftriplicate wells.

In vivo and ex vivo NIRF optical imagingNude mice bearing subcutaneous SKOV3-luc tumors were

intravenously injectedwith 4 nmol/L Cy5.5 fluorescent-labelednanoparticles and OA02-NPs, respectively. At different timepoint (0.5, 2, 4, 8, and 24 hours) postinjection, mice werescanned with Kodak imaging system IS2000MM. At 24 hours,tumors and major organs were excised for ex vivo imaging. Forthe microscopic analysis, excised tumors were frozen in

Xiao et al.

Cancer Res; 72(8) April 15, 2012 Cancer Research2102




optimum cutting temperature (OCT) medium at 80�C. Thecorresponding slices (10 mm) were prepared, air dried for 10minutes, and fixed with 4% paraformaldehyde for 10 min-utes. The a-3 integrin expression in the tumor section wasstained by Alex Fluor 488–conjugated a-3 integrin antibody(1:500) for 1 hour at room temperature. The blood vessel wasstained by rat anti-mouse CD31 primary antibody (Millipore,1:100) for 1 hour and Cy3-conjugated goat anti-rat IgGsecondary antibody (Millipore, 1:1,500) for 1 hour at roomtemperature.

Therapeutic studyThe antitumor efficacy and toxicity profiles of different

paclitaxel formulations were evaluated in the subcutane-ous xenograft mouse model of SKOV-3 ovarian cancer. Thetreatment was initiated when tumor volume reached 100 to200 mm3 and this day was designated as day 0. Themaximum tolerated dose (MTD) of Taxol in mice is approx-imately 10 mg/kg (6, 33), and the micellar formulations ofpaclitaxel were expected to be better tolerated than Taxolaccording to our previous report (6). Mice were adminis-

trated intravenously with PBS, Taxol (10 mg/kg), PTX-NPs(10, 30 mg/kg), and PTX-OA02-NPs (10, 30 mg/kg), respec-tively (n ¼ 8–10). The dosage was given every 3 days for atotal of 6 doses. Tumor sizes were measured with a digitalcaliper twice per week. Tumor volume was calculated bythe formula (L � W2)/2, where L is the longest and W isthe shortest in tumor diameters (mm). Relative tumorvolume (RTV) equals the tumor volume at given time pointdivided by the tumor volume before initial treatment. Forhumane reasons, animals were sacrificed when theimplanted tumor volume reached 1,500 mm3, which wasconsidered as the end point of survival data. Survival ratewas analyzed using a Kaplan–Meier plot. The potentialtoxicities after treatment were monitored by the animalbehavior observation and the body weight measurementtwice per week.

Statistical analysisStatistical analysis was conducted by the Student t test for

comparison of 2 groups, and one-way ANOVA for multiplegroups, followed by Newman–Keuls test if overall P < 0.05.

Figure 1. Chemical structure ofOA02 peptide functionalized PEG5K-CA8 telodendrimer (A) and thepreparation of stealth-targetednanoparticles by the self-assemblyof blank/OA02-functionalizedtelodendrimers (B). Alkyne-containing OA02 peptide wasconjugated to the azide group on thedistal terminus of PEG chain inPEG5K-CA8 telodendrimer via "clickchemistry". The Dde-protectedamino group on the proximal lysine inOA02-PEG5K-CA8 telodendrimercan be removed and used for theconjugation of fluorescence dyessuch as FITC or Cy5.5 for cell andanimal imaging. The OA02 peptidespresented on the surface of targetednanoparticles are able to specificallyrecognize and bind the a-3 integrinreceptors, which are overexpressedon the cell membrane of ovariancancer cells.






Results and DiscussionSynthesis of OA02-PEG5k-CA8 telodendrimer

N3-PEG5k-CA8 telodendrimer was first synthesized via

stepwise solution-phase condensation reactions as reportedpreviously (6). "Click chemistry" was used to covalentlyconjugate alkyne-containing OA02 peptide onto the azidegroup at the PEG terminus of N3-PEG

5k-CA8 telodendrimer,resulting in OA02-PEG5k-CA8 telodendrimer (Fig. 1A). The

conjugation of OA02 peptide onto PEG5k-CA8 telodendrimerwas confirmed by AAA (Supplementary Fig. S1). Each aminoacid of OA02-PEG5k-CA8 telodendrimer was hydrolyzed andquantitatively measured by HPLC. As summarized in Sup-plementary Table S1, the determined numbers of eachamino acid by AAA were almost identical to their corre-sponding theoretical values in the molecular formula ofOA02-PEG5k-CA8 telodendrimer, indicating the successful

Table 1. The physicochemical characteristics of PEG5K-CA8 and OA02-PEG5K-CA8 telodendrimers

TelodendrimersCMC,a

mmol/LParticlesize,b nm

Paclitaxel/polymerratio,c w/w

Paclitaxelloaded inmicelles,mg/mL EE (%)d

Particle sizeafter paclitaxelloading,b nm

Zetapotential, mV

PEG5K-CA8 6.1 21.0 � 2.3 1:4 4.6 � 0.5 92 � 1.8 46.2 � 3.4 �1.62OA02-PEG5K-CA8 11.8 20.5 � 1.9 1:4 4.8 � 0.8 96 � 2.7 49.1 � 2.8 �0.64

aCMC was measured by fluorescence spectrometry using pyrene (2 mmol/L) as a probe.bMeasured by DLS.cThe concentrations of telodendrimers were 20 mg/mL.dEncapsulation efficiency expressed as a percentage mean of 3 determinations � SD of paclitaxel weight recovered in nanoparticlescompared with theoretical loaded weight.

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Figure 2. The morphology (A) andparticle size distribution (B) of PTX-OA02-NPs (5 mg paclitaxel in 20mg/mL telodendrimer) measuredby cryo-TEM and DLS,respectively. A, white arrows pointto nanoparticles, while tobaccomosaic virus (TMV) was used ascalibration standard (18 nm inwidth). The ice crystals usuallyhave clear edge contrast. C, in vitropaclitaxel release kinetics fromPTX-NPs or PTX-OA02-NPs inPBS at 37�C. The concentration ofpaclitaxel remained in the dialysiscartridge at various timepointswasmeasured by HPLC. Error barswere obtained from triplicatesamples.

Xiao et al.





conjugation of OA02 peptide to the telodendrimer. Themolar ratio of OA02 peptide to telodendrimer was almost1:1, which meant that there was approximately one targetingpeptide molecule per telodendrimer monomer. The molec-ular weight of OA02-PEG5k-CA8 telodendrimer was mea-sured with MALDI-TOF MS. The monodispersed mass tracewas detected, and its molecular weight from MALDI-TOFMS was almost identical to the theoretical value (Supple-mentary Fig. S2A). The chemical structure of OA02-PEG5k-CA8 telodendrimer was also determined by 1H-NMR spec-trometry. As shown in Supplementary Fig. S2B, the signals at0.6 to 1.3 and 3.5 to 3.7 ppm could be assigned to cholic acidsand PEG chains, respectively. The 1H-NMR signals of OA02peptide were overlapped with the signals from the teloden-drimers, and no distinguishable signal was observed.

Preparation and characterization of paclitaxel-loadedOA02-PEG5k-CA8 nanoparticlesThe critical micelle concentration (CMC) of OA02-PEG5k-

CA8 telodendrimer was found to be comparable with that ofblank PEG5k-CA8 telodendrimer, with the range of 6 to 12mmol/L (Table 1 and Supplementary Fig. S3), indicating its

excellent micelle-forming property. Using the dry-down meth-od, hydrophobic drugs such as paclitaxel can be readily encap-sulated into the core of micellar nanoparticles. When thefeeding ratio of drug paclitaxel/telodendrimer (w/w) was1:4, the encapsulation efficiency of PEG5k-CA8 nanoparticlesand OA02-PEG5k-CA8 nanoparticles were approximately 92%and 96%, respectively. To enable the developed nanoparticlesto possess both antibiofouling ("stealth") and cell-specific–targeting properties (34), OA02-PEG5k-CA8 telodendrimer wasmixed with blank PEG5k-CA8 telodendrimer (1:1, w/w) toprepare PTX-OA02-NPs for the subsequent cell and animalstudies (Fig. 1B). The cryo-TEM image (Fig. 2A) showed thatPTX-OA02-NPs were spherical, with uniform particle sizes ofaround 50 nm in diameter, which was similar with the resultobtained from DLS measurement (Fig. 2B). The zeta potentialof PTX-OA02-NPs in PBS was almost neutral (�0.64 mV). Thestability of PTX-OA02-NPs was evaluated by measuring thechanges of particle sizes over time at different conditions. PTX-OA02-NPs were found to be very stable at 4�C for more than 3months, and also stable at 37�C for at least 72 hours whenincubated with 50% FBS (data not shown), which indicatedthat they will likely be able to maintain their stability and

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Figure 3. The uptake of FITC-labeled OA02-NPs in SKOV-3 ovarian cancer cells and K562 leukemia cells (a-3 integrin negative). A, confocal microscopicimages of SKOV-3 cell incubated with FITC-labeled nanoparticles and OA02-NPs (green) for 2 hours. To show the a-3 integrin-dependentuptake, excess amount of a-3 integrin antibody, or free OA02 peptide was added before the incubation of OA02-NPs with SKOV-3 cells. The flowcytometric analysis of OA02-NPs uptake in K562 cells (B) and SKOV-3 cells (C). The MFI of cells incubated with nanoparticles and OA02-NPsare shown as insets. �, P < 0.05. D, the addition of free OA02 peptides at concentrations from 2 to 200 mmol/L inhibited cellular uptake of OA02-NPs inSKOV-3 cells in a dose-dependent manner.






integrity during their in vivo applications. The drug releasepattern from PTX-OA02-NPs was similar with that from PTX-NPs, which both were biphasic, with the initial rapid release ofpaclitaxel during the first 4 hours, followed by the slow linearrelease over the subsequent few days (Fig. 2C). In summary, thedecoration of OA02 peptide had negligible impact on thephysicochemical properties of PEG5k-CA8 nanoparticles,including CMC, morphology, particle size, drug-loading capac-ity, encapsulation efficiency, stability, and drug release profile.This is probably because the OA02 peptide conjugation occursat the distal end of PEG chain which is located on the shell ofself-assembled micellar nanoparticles without interfering withthe drug-holding hydrophobic core unit.

Cellular uptake studiesa-3 Integrinwas showed to be overexpressed on both SKOV-

3 and ES-2 cells, as measured by the flow cytometric analysis

(Supplementary Fig. S4). The uptake profiles of FITC-labeledOA02-NPs in ovarian cancer cells were first qualitativelyobserved by the confocal microscopy. Nontargeted nanopar-ticles had minimal nonspecific cellular uptake after 2-hourincubation, whereas the decoration of OA02 peptide greatlyincreased the extent of nanoparticles uptake in both SKOV-3(Fig. 3A) and ES-2 cells (Supplementary Fig. S5A). Most FTIC-labeled nanoparticles (green) distributed around the perinuc-lear region, which meant that these nanoparticles were inter-nalized into the cytoplasm. More importantly, the uptake ofOA02-NPs in both ovarian cancer cells was able to be remark-ably inhibited by excess amount ofa-3 integrin antibody or freeOA02 peptide, suggesting the a-3 integrin receptor-dependentinternalization of OA02-NPs.

The uptake efficiencies of FITC-labeledOA02-NPs in ovariancancer cells and K562 leukemia cells (a-3 integrin negative)were further quantitatively measured by the flow cytometricanalysis. Both nontargeted nanoparticles and OA02-NPs hadsimilar low nonspecific uptake in K562 cells (Fig. 3B). However,OA02-NPs exhibited significantly higher uptake than the non-targeted nanoparticles in both ovarian cancer cells (P < 0.05),with almost 6-fold higher uptake in SKOV-3 cells (Fig. 3C) and4-fold higher uptake in ES-2 cells (Supplementary Fig. S5B),respectively. The addition of free OA02 peptide was able toinhibit the uptake of OA02-NPs in SKOV-3 cells (Fig. 3D) andES-2 cells (Supplementary Fig. S5C) in a dose-dependentmanner, further confirming the a-3 integrin–targeting speci-ficity of OA02-NPs. It should be noted that significantly higherconcentration of free peptide (10- to 100-folds) was required toinhibit the cellular uptake of OA02-NPs, whereas the equalconcentration of free peptide did not produce obvious inhi-bition effect. This could be possibly explained by the increasedbinding affinity of OA02 peptides presented on the nanopar-ticles surface due to the multivalency effects (35).

Intracellular tracking of dual fluorescent-labeledOA02-NPs in live cells

After 2-hour incubation, both FITC-labeled telodendrimercarrier (green) and DiD dye payload (red) were simultaneouslyinternalized into the SKOV-3 cells with colocalized dot-shapefluorescent foci in the perinuclear region of the cytoplasm (Fig.4A, bottom left), indicating that the intact OA02-NPs weretaken up into the cytoplasm. These fluorescent foci weregenerated as a result of the accumulation of OA02-NPs in theendocytic vesicles (i.e., endosomes and lysosomes). This wasevidenced by the partial colocalization of internalized FITC-labeled OA02-NPs (green) with lysosomal compartment (red),producing yellow fluorescence in the merge images (Fig. 4A,bottom right). Similar observationwas also reported byGu andcolleagues in the LNCaP prostate cancer cells incubatedwith A10 aptamer–targeted PLGA-b-PEG-b-Apt nanoparticles(34).

In vitro cytotoxicity studyPTX-OA02-NPs were found to be significantly more cyto-

toxic against both SKOV-3 (Fig. 4B) and ES-2 cells (Supple-mentary Fig. S6), when compared with nontargeted PTX-NPsat the equivalent paclitaxel concentration (P < 0.05). The

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Figure 4. A, intracellular tracking of dual fluorescent-labeled OA02-NPsupon their uptake in live SKOV-3 cells. DiD dye (red) was encapsulated asdrug surrogates into FITC (green) covalently labeled OA02-NPs. SKOV-3cells were incubated with DiD/FITC dual-labeled OA02-NPs for 2 hours,and the cells were stained with lysosome tracker (red) before the live-cellimaging by confocal microscopy. B, the differential cytotoxicities of 0.5mg/mL paclitaxel-loaded nanoparticles (PTX-NPs), paclitaxel-loadedOA02-NPs (PTX-OA02-NPs), and the equivalent dose of blanknanoparticles, OA02-NPs against SKOV-3 ovarian cancer cellsmeasured by MTT assay. Nanoparticles were incubated with cells for 2hours, and the cells were subsequently washed and incubated in freshmedia for a total of 72 hours before assessing cell viability in each group.�, P < 0.05.

Xiao et al.





enhanced cytotoxicity of PTX-OA02-NPs is likely related tothe capability of OA02 peptide to facilitate the uptake ofPTX-OA02-NPs into ovarian cancer cells, thus increasing theintracellular paclitaxel concentration. Furthermore, therewas no observable cytotoxic effect associated with bothblank nontargeted nanoparticles and OA02-NPs at the equiv-alent nanoparticles concentration, which eliminated thepossibility of OA02 peptides or nanoparticle-induced cyto-toxic activity.

Biodistribution and tumor targeting specificity in vivoNIRF optical imaging is an important tool for visualizing

molecular processes in vivo, as NIRF dyes with deep pene-tration, low autofluorescence, and low tissue absorption andscattering enable the high-resolution tissue imaging (26).Both Cy5.5 fluorescent-labeled nanoparticles and OA02-NPs

distributed throughout the body of the mice immediatelyafter the intravenous injection and gradually accumulatedinto the SKOV3-luc tumor. However, the uptake rate ofOA02-NPs in the tumor site was faster than that of non-targeted nanoparticles. Substantial contrast between tumorsand background in the mice injected with OA02-NPs wasobserved at around 4-hour postinjection, and these nano-particles were able to be retained in the tumor throughoutthe 24-hour period, whereas the accumulation of nontar-geted nanoparticles in the tumor was not apparent until 8-hour postinjection (Fig. 5A). Ex vivo images at 24 hoursshowed that both nontargeted nanoparticles and OA02-NPsexhibited relatively high uptake in the SKOV3-luc tumorthan in normal organs except the liver (macrophage uptake),as the result of EPR effect (Fig. 5B). However, the meanfluorescence intensity (MFI) of tumors for OA02-NPs was

Tumor

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Figure 5. In vivo and ex vivo NIRF optical imaging of Cy5.5-labeled nanoparticles or OA02-NPs biodistribution after i.v. injection in subcutaneous SKOV3-luctumor–bearing mice. A, in vivo optical images of real-time tumor-targeting characteristics of nanoparticles or OA02-NPs. Tumor-bearing mice were injectedvia tail vain with 4 nmol/L Cy5.5-labeled nanoparticles or OA02-NPs and were scanned with Kodak multimodal imaging system IS2000MM at different timepoint. B, representative ex vivo optical images of tumors and organs of SKOV3-luc tumor–bearing mice sacrificed at 24 hours. C, quantitative fluorescenceintensities of tumors and organs from ex vivo images (n ¼ 3). D, histologic analysis of nanoparticles or OA02-NPs distribution (Cy5.5, red) in tumorcryosections. The nuclei were stained byDAPI (blue),a-3 integrin expression onSKOV-3 tumor cells and vascular endothelial cells were visualized by anti-a-3(green) and anti-CD31 (orange) staining, respectively. AU, arbitrary units.






approximately 1.7-fold higher than that for nontargetednanoparticles (Fig. 5C, P < 0.05). The histologic distributionof nanoparticles in the tumor tissue was further observedunder the confocal microscopy. As shown in Fig. 5D, themajority of Cy5.5-labeled nontargeted nanoparticles (red)were mainly distributed in the perivascular region, which isin concordance with previous histologic observations ofpassive accumulation of liposomes and micelles in tumortissues (14). In contrast, OA02-NPs were able to extravasatefrom the tumor vasculature, penetrate deep into the inter-stitial space of the tumor, bind to a-3 integrin–overexpres-sing tumor cells, and eventually become internalized. Similarobservations were also reported by Kirpotin and colleaguesusing PEGylated liposome targeted by an anti-HER-2 anti-body (36). The enhanced tumor localization and intracellularuptake of OA02-targeted nanoparticles into ovarian cancercells is probably attributed to the specific interactionbetween the OA02 peptide and a-3 integrin, thus facilitatingthe homing of targeted nanoparticles to ovarian cancer, andthe receptor-mediated endocytosis.

Therapeutic studyThe antitumor effect of PTX-OA02-NPs was evaluated in the

subcutaneous SKOV-3 ovarian cancer xenograft mouse modelwhen compared with nontargeted PTX-NPs and paclitaxelclinical formation (Taxol). As shown in Fig. 6A, all the paclitaxelformulations significantly inhibited the tumor growth, whencompared with the control group (P < 0.05). However, bothPTX-NPs and PTX-OA02-NPs exhibited better tumor growthinhibition than Taxol, at equivalent paclitaxel dose of 10mg/kg. The superior tumor growth inhibition of paclitaxelmicellar formulationsmight be attributed to the larger amountof paclitaxel delivered to the tumor site via the EPR effect.Moreimportantly, mice treatedwith PTX-OA02-NPs had significant-ly slower tumor growth rate than those treated with nontar-geted PTX-NPs at the equivalent paclitaxel doses (P< 0.05). Thesurvival rate of mice in each group is presented by the Kaplan–Meier survival curve, respectively (Fig. 6B). In general,compared with PBS control, all the paclitaxel formulationssignificantly prolonged the survival rates of tumor-bearingmice. However, mice treated with 30 mg/kg PTX-OA02-NPs

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Figure 6. In vivo tumor growth inhibition (A), Kaplan–Meier survival curves (B), and body weight changes (C) of SKOV-3 tumor–bearing mice after theintravenous treatment of various paclitaxel formulations. Tumor-bearingmicewere administered intravenouslywith PBS (control), Taxol (10mg/kg), PTX-NPs(10 and30mg/kg), andOA02-PTX-NPs (10 and30mg/kg), respectively, every 3 daysondays 0, 3, 6, 9, 12, and15 for a total of 6 doses. Data representmean�SEM (n ¼ 8–10).

Xiao et al.





exhibited the longest survival time among these treatmentgroups. The median survival time of mice in the group of PBScontrol, Taxol, PTX-NPs (10, 30 mg/kg), and PTX-OA02-NPs(10, 30mg/kg)were 20, 27, 29, 69, 32, and 95 days, respectively. Itwas also noted that complete response (CR), defined as acomplete disappearance of palpable tumor nodule, wasachieved in 5 of 10 mice (50%) in the 30 mg/kg PTX-OA02-NPs group, and 2 of 8 mice (25%) in the 30 mg/kg PTX-NPsgroup. The better tumor growth inhibition, prolonged survivaltime, and higher complete tumor response rate observed in thePTX-OA02-NPs group are likely due to the more efficient andspecific delivery of paclitaxel to the ovarian cancer cells by theunique ligand-targeted nanoformulation, asmentioned earlier.Several independent investigations have also showed thatsome other targeted nanoparticles such as antibody-targetedliposomes (37), transferrin-targeted polymeric nanoparticles(38), and folate-targeted polyamidoamine (PAMAM) polymers(39) can significantly enhance the antitumor effects as com-pared with their nontargeted counterparts.Toxicities were assessed by direct observation of animal

behavior and body weight monitoring. Mice treated with 10mg/kgTaxol showed adecline of overall activity during thefirst30-minute postinjection, which is likely a sign of hypersensi-tivity reaction related to the diluent (Cremophor EL andethanol; ref. 33). In addition, this group of mice was found tohave significant amount of body weight loss (with a nadir of6.7%) after receiving the first few doses, when compared withthe control group (P < 0.05). In contrast, the mice in both PTX-NPs and PTX-OA02-NPs groups tolerated the regimens well.The treatments did not seem to have obvious adverse impacton their activity level and body weight (Fig. 6C). Overall, thepaclitaxel nanoformulations, both PTX-NPs and PTX-OA02-NPs, showed with an improved systemic toxicity profile, whichmight be attributed to their Cremophor-free composition,prolonged blood retention time, and sustained drug releasefeatures (40).

ConclusionWe have successfully developed OA02 peptide–targeted

polymeric micelles system to effectively deliver chemothera-peutic drugs to ovarian cancer. High-affinity and high-speci-

ficity "OA02" peptide against a-3 integrin was successfullyconjugated to the distal PEG terminus of PEG5k-CA8 teloden-drimer via "click chemistry" and displayed on the surface ofself-assembled micellar nanoparticles. OA02 peptide decora-tion dramatically enhanced the intracellular delivery of nano-particle drugs intoa-3 integrin–overexpressing ovarian cancercells via receptor-mediated endocytosis, resulting in higher invitro cytotoxicity of PTX-OA02-NPs against those cancer cellsthan nontargeted PTX-NPs. OA02 peptide also significantlyfacilitated the distribution of targeted nanoparticles intotumor tissues and cells in SKOV3-luc tumor–bearing mice.Furthermore, PTX-OA02-NPs were found to be more effica-cious and less toxic than the equivalent doses of nontargetedPTX-NPs and Taxol in ovarian cancer xenograft mouse model.Therefore, OA02 peptide–targeted nanoparticles drug deliverysystem has great translational potential in the treatment ofpatients with ovarian cancer.

Disclosure of Potential Conflicts of InterestK.S. Lam is the founding scientist of LamnoTherapeutics which plan to

develop the nanotherapeutics described in the manuscript. J. Luo and K. S. Lamare the inventors of pending patent on telodendrimers. J.S. Lee has receivedCommercial Research Grant from Hyundai Hope on Wheel Pediatric CancerResearch Grant.

Authors' ContributionsConception and design: K. Xiao, Y. Li, R.H. Cheng, J. Luo, K.S. LamDevelopment of methodology: K. Xiao, Y. Li, J. LuoAcquisition of data: K. Xiao, Y. Li, A.M. Gonik, E. Sanchez, L. Xing, R.H. ChengAnalysis and interpretation of data: K. Xiao, Y. Li, J.S. Lee, A.M. Gonik, R.H.Cheng, J. Luo, K.S. LamWriting, review, and/or revision of themanuscript: K. Xiao, Y. Li, J.S. Lee, A.M. Gonik, J. Luo, K.S. LamAdministrative, technical, or material support: J.S. Lee, T. DongStudy supervision: K.S. Lam

Grant SupportThis study was supported by NIH/NCI R01CA140449 (to J. Luo), R01CA115483

(to K.S. Lam), R01EB012569 (to K.S. Lam), and DoD Postdoctoral FellowshipAward (W81XWH-10-1-0817; to K. Xiao).

The costs of publication of this article were defrayed in part 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 November 28, 2011; revised February 3, 2012; accepted February 19,2012; published OnlineFirst March 6, 2012.

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2012;72:2100-2110. Published OnlineFirst March 6, 2012.Cancer Res Kai Xiao, Yuanpei Li, Joyce S. Lee, et al.

In VivoPaclitaxel-Loaded Micellar Nanoparticles to Ovarian Cancer ''OA02'' Peptide Facilitates the Precise Targeting of

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