Receptor-Targeted Nanoparticles for In vivo Imaging of

Receptor-Targeted Nanoparticles for In vivo Imagingof Breast CancerLily Yang,1,2,4 Xiang-Hong Peng,1Y. Andrew Wang,6 Xiaoxia Wang,2 Zehong Cao,1Chunchun Ni,2

PrasanthiKarna,1XinjianZhang,5 WilliamC.Wood,1,4XiaohuGao,3 ShumingNie,3,4 andHuiMao2,4

Abstract Purpose: Cell-surface receptor-targeted magnetic iron oxide nanoparticles provide molecularmagnetic resonance imaging contrast agents for improving specificity of the detection of humancancer.Experimental Design:The present study reports the development of a novel targeted ironoxidenanoparticle using a recombinant peptide containing the amino-terminal fragment of urokinase-typeplasminogenactivator (uPA) conjugated tomagnetic ironoxidenanoparticles amino-terminalfragment conjugated-ironoxide (ATF-IO).This nanoparticle targets uPA receptor, which is overex-pressedinbreastcancer tissues.Results: ATF-IO nanoparticles are able to specifically bind to and be internalized by uPAreceptor ^ expressing tumor cells. Systemic delivery of ATF-IO nanoparticles into mice bearings.c. and i.p. mammary tumors leads to the accumulation of the particles in tumors, generating astrongmagnetic resonance imaging contrast detectable by a clinicalmagnetic resonance imagingscanner at a field strength of 3 tesla.Target specificity of ATF-IO nanoparticles showed by in vivomagnetic resonance imaging is further confirmed by near-IR fluorescence imaging of themammary tumors usingnear-IR dye-labeled amino-terminal fragment peptides conjugated to ironoxide nanoparticles. Furthermore, mice administered ATF-IO nanoparticles exhibit lower uptakeof the particles in the liver and spleen compared with those receiving nontargeted iron oxidenanoparticles.Conclusions: Our results suggest that uPA receptor ^ targeted ATF-IO nanoparticles havepotential as molecularly targeted, dual modality imaging agents for in vivo imaging of breastcancer.

Breast cancer is themost common type of cancer and the secondleading cause of cancer-related death among women. Novelapproaches for the detection of primary and metastatic breastcancers are urgently needed to increase the survival of patients. Apromising strategy to improve the specificity and sensitivity ofcancer imaging is to use biomarker target–specific imagingprobes (1–3) for image-based diagnosis and treatment moni-toring. Currently, targeted radionuclide probes have been usedfor cancer detection by positron emission tomography or singlephoton emission tomography (2, 4, 5). Although nuclearimaging modalities show a high sensitivity, they lack goodresolution and anatomic localization of the tumor lesion andrequire complicated and expensive radiochemistry. In addition,the half-life of the radiotracer often limits the ability for dynamicand time-resolved imaging and may not be able to capture thebiomarker-targeting agent to reach and accumulate in the tumor.Magnetic resonance imaging offers a high spatial resolution andthree-dimensional anatomic details and has been widely used inclinical oncology imaging. Recently, breast magnetic resonanceimaging was recommended by the American Cancer Society as ascreening approach, adjunct to mammography, for the earlydetection of breast cancer in women at high risk for this disease(6). Although breast cancermagnetic resonance imaging shows a

Imaging, Diagnosis, Prognosis

Authors’ Affiliations: Departments of 1Surgery, 2Radiology, and 3BiomedicalEngineering, 4Winship Cancer Institute, Emory University School of Medicine;5Department of Chemistry, Georgia State University, Atlanta, Georgia; and 6OceanNanotech, LLC, Springdale, ArkansasReceived12/22/08; revised3/6/09; accepted3/27/09;publishedOnlineFirst7/7/09.Grant support: Emory-GeorgiaTech Nanotechnology Center for Personalized andPredictive Oncology of the NIHNational Cancer Institute (NCI) Center of CancerNanotechnology Excellence (U54 CA119338), NIHNCIR01CA133722 (L.Yang),EmoryMolecular andTranslational Imaging Center grant (NIH, NCI, P50CA128613),Seed Grant from EmTech Bio, Inc. (H. Mao), the Friends forAn Early Breast CancerTest Foundation, the Idea Award of the Breast Cancer Research Program of theDepartmentofDefense (BC021952), and theNancyPanozEndowedChair (L.Yang).The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18U.S.C.Section1734 solely to indicate this fact.Note:L.YangandX-H.Pengcontributedequally to the studies in this article.Current address forX.Gao:DepartmentofBioengineering,UniversityofWashington,Seattle,Washington.Requests for reprints: Lily Yang, Department of Surgery andWinship CancerInstitute, Emory University School of Medicine, C-4088, 1365 C Clifton Road NE,Atlanta, GA 30322. Phone: 404-778-4269; Fax : 404-778-5530; E-mail:[email protected] or Hui Mao, Department of Radiology, Emory Center forSystems Imaging, Emory University School of Medicine, 1841 Clifton Road,Atlanta, GA 30329. Phone: 404-712-0357; Fax : 404-712-5948; E-mail:[email protected].

F2009 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-08-3289

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high sensitivity in detecting small breast lesions, a majorchallenge is its low specificity when using a nontargeted contrastagent such as gadolinium chelates, resulting in a high false-positive rate and unnecessary biopsy and mastectomy (7).Therefore, the development of molecularly targeted magneticresonance imaging contrast agents may increase the specificity ofmagnetic resonance imaging, as well as provide information onthe level of biomarker expression in breast cancers.

Recently, biocompatible and functionalized nanoparticleshave been shown to target tumors and produce optical,magnetic, and/or radioactive signals for enhancing sensitivityand specificity of noninvasive tumor imaging. Previous studieshave shown the feasibility of producing such imaging probes forin vivo magnetic resonance imaging of cancers (8–10). Theunique features of nanoparticles that make them suitable forreceptor-targeted imaging include (a) having a prolonged bloodretention time and (b) providing reactive function groups and alarge surface area for loading large numbers or multiple types oftumor-targeting ligands. However, several issues remain to beaddressed in making nanoparticle imaging probes for clinicalapplications. These include the identification and availability ofsuitable imaging biomarkers, the delivery of sufficient amount ofprobe in vivo, and the development of imaging probes withsufficient signal amplification and contrast enhancement (11).

Paramagnetic iron oxide nanoparticles can induce remarkablystrong magnetic resonance imaging contrast and have a largesurface area and versatile surface chemistry for surface function-alization and the introduction of biomolecules (12–14). Theirbiological safety in humans has been tested, with nontargetediron oxide nanoparticles currently in use to detect liver tumorlesions or lymph node metastases in patients (15, 16). Previousattempts to develop targeted iron oxide nanoparticles useddextran or polyethylene glycol–coated iron oxide to conjugatetargeting ligands and showed the feasibility and improvedsensitivity and specificity of such biomarker-targeted magneticresonance imaging nanoprobes (3, 14, 17–19). However, mostof these ligands were directed to molecular targets that were

expressed only in a small percentage of tumor tissues orsubpopulations of tumor cells, such as HER-2/neu, transferrin,mucin 1, and folate acid, which limits the sensitivity and ap-plication for molecularly targeted cancer imaging in patients.

In this study, we used the amino-terminal fragment of the highaffinity receptor binding domain of urokinase-type plasminogenactivator (uPA) to target its cellular receptor, which is up-regulated in a high percentage of tumor cells and tumor-associated stromal cells, such as endothelial cells, macrophages,and fibroblasts, of many human cancer types (20–23). It is wellknown that the interaction of uPA with its cellular receptorresults in conversion of plasminogen to serine protease, a centralregulator of the activation of other proteases, including matrixmetalloproteinase, which promotes tumor metastasis andangiogenesis. An elevated level of uPA receptor is associatedwith tumor aggressiveness, the presence of distant metastasis,and poor prognosis in breast cancer patients (24–28). In humanbreast cancer tissues, high levels of uPA receptor are detected in54% of ductal carcinoma in situ and in 60% to 90% of invasivebreast cancer tissues (24, 29).

To achieve optimal tumor targeting and imaging, we havedeveloped novel paramagnetic iron oxide nanoparticles that haveuniform core sizes and are functionalized through surface coatingof ampiphilic polymers. This surface coating provides a stablehydrophobic protective inner layer around a single crystal of ironoxide nanoparticle with carboxylate groups in the outer layerreadily available for conjugation with amino-terminal fragmentpeptides. Amino-terminal fragment conjugated-iron oxide(ATF-IO) nanoparticles specifically bind to and are internalizedby uPA receptor–expressing cells. We showed that systemicdelivery of ATF-IO nanoparticles into mice bearing mammarytumors led to the accumulation of the targeted nanoparticles insubcutaneous (s.c.) and metastatic mammary tumors, inducingmagnetic resonance imaging contrast changes sufficient for in vivotumor imaging. By conjugation of Cy5.5, a near-IR dye, to theamino-terminal fragment peptides, wewere able to achieve in vivotumor imaging with magnetic resonance imaging and opticalimaging.

Materials and Methods

Breast cancer cell lines

Mouse mammary carcinoma cell line 4T1 stably expressing a fireflyluciferase gene was obtained from Dr. Mark W. Dewhirst at DukeUniversity (Durham, NC). Human breast cancer cell line T47D waspurchased from American Type Culture Collection.

Preparation of amino-terminal fragment–iron oxide

nanoparticles

A cDNA fragment encoding amino acids 1 to 135 of mouse uPA,isolated by PCR amplification using a PCR primer pair containingforward (5¶-CACCATGGGCAGTGTACTTGGAGCTCC-3¶) and reverse (5¶-GCTAAGAGAGCAGTCA-3¶) primers, was cloned into pET101/D-TOPOexpression vector (Invitrogen). Recombinant amino-terminal fragmentpeptides were expressed in Escherichia coli BL21 (Invitrogen) and purifiedfrom bacterial extracts under native conditions using a Ni2+ nitrilotri-acetic acid (NTA)-agarose column (Qiagen). Purification efficiency wasdetermined by SDS-PAGE and >95% of purified proteins were amino-terminal fragment peptides. Cy5.5 maleimide (GEHealthcare), a near-IRdye, was conjugated to amino-terminal fragment peptides using themanufacturer’s protocol. Nontargeted dye molecules were separatedfrom the Cy5.5 dye– labeled amino-terminal fragment peptides usingSephadex G25 column.

Translational Relevance

Breast cancer is themostcommoncancer inwomenwithabout180,000 new cases and 40,000 deaths each year inthe United States. Currently, breast magnetic resonanceimaging is used for early detection of breast cancer and isrecommended as a routine screen for women at high riskfor the disease. Althoughbreast magnetic resonance imag-ing using a nontargeted contrast agent has a relatively highsensitivity in detecting lesions, it lacks specificity in deter-mining the pathologic characteristics of the lesions. Recentstudies show that patients receiving magnetic resonanceimaginghave ahigher rate of biopsy andmastectomy com-paredwith thosewithout this imagingprocedure.Magneticresonance imaging is a promising noninvasive imagingapproach for preoperative staging of breast cancer andmonitoring tumor response to therapy.Therefore, thedevel-opment of biomarker-targeted magnetic resonance imag-ing contrast probes that increase the specificity of breastcancer magnetic resonance imaging should have a greatimpact on the detection and treatment of breast cancer.

Receptor-Targeted Nanoparticles forTumor Imaging

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Paramagnetic iron oxide nanoparticles were prepared as described(30) using iron oxide powder as the iron precursor, oleic acid as theligand, and octadecene as the solvent. The core size and hydrodynamicsize of the iron oxide nanoparticles were measured using transmissionelectron microscopy and light scattering scan, respectively. The particleswere coated with amphiphilic polymers using a similar method, asreported previously (31). Amino-terminal fragment peptides wereconjugated to the surface of iron oxide nanoparticles via cross-linkingof carboxyl groups to amino side groups on the amino-terminal fragmentpeptides, as shown in Fig. 1. Briefly, the polymer-coated iron oxide nan-oparticles were activated with ethyl-3-dimethyl amino propyl carbodii-mide (Pierce) and sulfo-N-hydroxysuccinimide for 15 min. Afterpurification using Nanosep 100k OMEGA (Pall Corp.), activated ironoxide nanoparticles were reacted with amino-terminal fragment orCy5.5–amino-terminal fragment peptides at a molar ratio iron oxide toamino-terminal fragment of 1:20 in PBS (pH 7.0) at 4jC overnight,generating amino-terminal fragment– iron oxide or Cy5.5–amino-terminal fragment–iron oxide nanoparticles. The final amino-terminalfragment– iron oxide conjugates were purified using Nanosep 100kcolumn filtration. Conjugation efficiency of amino-terminal fragmentpeptides to the iron oxide nanoparticles was confirmed by the mea-surement of fluorescence intensity of Cy5.5–amino-terminal fragment–iron oxide nanoparticles using fluorescence spectroscopy and the fpotential change before and after conjugation of the amino-terminalfragment ligands to iron oxide nanoparticles. The number of amino-terminal fragment peptides conjugated to each iron oxide nanoparticlewas estimated by measuring the fluorescence intensity of a dilutedsample of Cy5.5–amino-terminal fragment–iron oxide nanoparticlesusing an emission wavelength of 696 nm and then comparing the valueto a linear standard curve prepared using various concentrations ofCy5.5–amino-terminal fragment peptides.

Western blot analysis

Western blot analysis was done as described using a standard protocolin our laboratory (32). To confirm the presence of amino-terminalfragment peptides in SDS/PAGE gel, the protein was transferred topolyvinylidene difluoride membranes (Bio-Rad laboratories). An anti–His tag monoclonal antibody (Novagen) was used to identify the His-tagged amino-terminal fragment-peptides. After reacting with horserad-ish peroxidase– labeled rat anti–mouse immunoglobulin G antibody,the amino-terminal fragment peptide band was detected by enhancedchemiluminescence using ECL Plus (GE Healthcare) followed byautoradiography. The level of uPA receptor was determined using ananti–uPA receptor polyclonal rabbit antibody (Santa Cruz Biotechnol-ogy) that reacts with mouse and human uPA receptor and horseradishperoxidase–conjugated goat anti – rabbit immunoglobulin G. Theprotein bands were detected using ECL.

Ni-NTA-agarose pull-down assays

To determine whether the purified recombinant amino-terminalfragment peptides, either free or conjugated to iron oxide nanoparticles,bind to uPA receptor, we did a combined pull-down and Western blotanalysis. Ni2+-NTA-agarose beads were incubated with His-taggedamino-terminal fragment peptides or amino-terminal fragment– ironoxide nanoparticles at 4jC for 30 min. The conjugated beads werewashed twice with binding buffer and incubated with 500 Ag of total celllysate obtained from 4T1 or T47D cells for 2 h. Bound proteins wereeluted from the beads using elution buffer containing 400 mmol/Limidazole and then examined by Western Blot analysis to determine theamount of uPA receptor pulled down by amino-terminal fragment– oramino-terminal fragment– iron oxide–conjugated Ni-NTA agarosebeads in each sample, as described above.

Fig. 1. Characteristics ofATF-IOnanoparticles.Uniform-size IOnanoparticleshave a10-nmcore size as shownby transmissionelectronmicroscopyandare coatedwithamphiphilic copolymersmodifiedwith shortpolyethylene glycol chains (scalebar: 50nm).Thepurityof recombinant amino-terminal fragment (ATF)peptideswas confirmedbyCoomassieblue stainingofSDS-PAGEgelandbyWesternblottingusingananti ^ His tagantibody.Cy5.5maleimidewas conjugated toATFpeptides throughabondbetweenmaleimide esters and free thiolgroupsof cystidine residuesof theATFpeptide.ATForCy5.5^ ATFpeptideswere thenconjugated to IOnanoparticles via the carboxylgroupsmediatedbyethyl-3-dimethylaminopropylcarbodiimide (EDAC).





Immunofluorescence labeling

Acetone-fixed frozen normal breast and cancer tissue sections wereincubated with 5 Ag/mL of polyclonal rabbit anti –uPA receptorantibody, followed by biotinylated goat anti– rabbit immunoglobulinG. After further incubation with Texas-red avidin, the slides wereexamined under a fluorescence microscope (Zeiss Axioplan withAxiovision software, Carl Zeiss MicroImaging, Inc.). To detect the levelof uPA receptor expression in living cancer cells, cells were incubatedwithan anti–uPA receptor antibody at 4jC for 30 min. After incubation withFITC–goat anti–rabbit immunoglobulin G, cells were examined underthe fluorescence microscope.

Specificity of ATF-IO nanoparticles to cancer cells

Cells cultured on glass chamber slides (Nalge Nunc International)were incubated with 13.5 pmol of Cy 5.5–ATF-IO or nontargeted ironoxide nanoparticles at 37jC for 3 h. After washing with PBS, the slideswere examined under a confocal microscope (Perkin Elmer UltraviewERS, PerkinElmer Life and Analytic Sciences, Inc.). To localize the ironoxide nanoparticles, the cells incubated with Cy5.5–ATF-IO or ironoxide nanoparticles were fixedwith 4% formaldehyde in PBS and stainedwith Prussian blue staining by incubating the cells with a 1:1 mixture of5% potassium ferrocyanide and 5% HCl acid for 30 min at 37jC toconfirm the presence of iron oxide nanoparticles (33).

In vitro magnetic resonance imaging scan

Cells were incubated with serum-freemedium containing nontargetediron oxide or ATF-IO nanoparticles at 37jC for 3 h. After washing withPBS, the cells were embedded in 0.8% agarose in 24-well plates. Plateswere then scanned in a 3-T magnetic resonance imaging scanner (PhilipsMedical System) using a T1-weighted gradient echo sequence and amulti-echo T2–weighted fast spin echo sequence, which simultaneouslycollects a series of data points at different echo times (i.e., 20 echo time(TE) points from 10-200 ms with a 10 ms interval) for T2 relaxometrymeasurement.

Mouse mammary tumor models

S.c. tumor model. Mousemammary tumor 4T1 cells were injected s.c.into the back flank area of 6- to 8-wk-old female BALB/c or nudemice.Weused nude mice for optical imaging to reduce background fluorescence.Intraperitoneal (i.p.) metastatic mammary tumor model. 4T1 cells

stably transfected with a firefly luciferase gene were directly injected intothe upper right side of the peritoneal cavity. Tumor growth wasmonitored by bioluminescence imaging using the Xenogen biolumines-cence imaging system (Xenogen Corp.).

In vivo magnetic resonance imaging of mouse mammary tumors.

Tumor-bearing mice were examined using a 3-T magnetic resonanceimaging scanner (Philips Medical System), with a customized rodentcoil to obtain magnetic resonance images. Because magnetic resonanceimaging contrast effect is greatly dependent on the magnetic fieldstrength (34), we chose to test the feasibility of detecting iron oxidenanoparticle– induced contrast at a clinically relevant field strength(3 T), with consideration of the potential application of thisimaging probe in patients. Animals were anesthetized by i.p.injection of a ketamine-xylazine mixture (95:5 mg/kg). Animalswere kept warm in the scanner using warm pads. A set of surveyimages was obtained using T2-weighted fast spin echo imagingsequence with repetition time (TR) of 5,000 ms and TE of 120 ms.This was followed by high resolution imaging in the coronal view(i.e., slices cut through from head to tail), with a field of view of110 � 40 mm, imaging matrix of 256 � 192 (reconstructed to356 � 256), and 40 slices with 1.1-mm slice thickness withoutslice gap.

The imaging sequences included T1- and T2-weighted spin echo orgradient echo methods and the three-dimensional fast-spoiled gradientecho technique. A TE of 10 ms and TR of 350 ms were used for T1-weighted spin echo imaging, and TE of 50 ms and TR of 1,500 ms wereused for T2-weighted fast spin echo imaging. A multi-echo T2 weighted

fast spin echo sequence with 12 TEs (range, 10-120 ms; a 10 ms interval)was used to obtain T2 maps of the whole mouse. The mice were injectedwith various nanoparticles suspended in PBS, through the tail vein andthen scanned at different time points. For postcontrast scan, care wastaken to maintain the same animal positions and use same imagingsequences and parameters across different scan sessions. Images frompre- and postcontrast administration were compared to evaluate theefficacy of contrast enhancement. Multi-echo T2 images of slices wereused for calculating T2 maps using a home-developed program based onMatlab (TheMathworks, Inc.). Because iron oxide nanoparticles typicallyinduced T2-weighted contrast change, the signal reduction in T2-weighted imaging and change in the T2 value were used to follow andestimate the accumulation of nontargeted or targeted iron oxidenanoparticles in the area. Region of interest analysis was used to evaluateand quantify the contrast agent– induced changes in magnetic resonanceimaging signal or T2 value in the tumor and other selected tissues andorgans. Regions of interest of tumors were drawn based on the tumor T2-signal enhancement in T2-weighted images or at a long TE timepoint (TE,80 ms) in multi-TE T2-mapping imaging.

Near-IR optical imaging of mouse mammary tumors

Tumor-bearing mice were placed on an alfalfa-free rodent diet (Teklad2918,Harlan Teklad) to reduce background fluorescence. Near-IR imagesof the tumor-bearingmice were taken using the Kodak in vivo FX imagingsystem (Carestream Molecular Imaging) before and at different timepoints after the nanoparticle injection. For each near-IR image, acorresponding X-ray image was taken to provide anatomic registrationof the tumor.

Histologic analysis

Tumor and normal tissues were collected from the mice at the end ofin vivo imaging experiments. Five-micrometer frozen tissue sectionswere incubated with Prussian blue staining solution to confirm thepresence of iron oxide nanoparticles in the tissue sections.

Results

Characterization of ATF-IO nanoparticles. The magnetic ironoxide nanoparticles synthesized for this study have a 10-nm coresize. We coated the nanoparticles with a monolayer ofamphiphilic polymers grafted with carbon alkyl side chains tostabilize and functionalize their surface, resulting in an estimated18-nm water-soluble iron oxide nanoparticle with carboxyl sidegroups. To reduce nonspecific binding and uptake by normaltissues, short polyethylene glycol chains were conjugated to aportion of the carboxyl side groups on the amphiphilicpolymers. Our results showed that magnetic iron oxide nano-crystal used in this study has a strong T1- and T2-shortening effectwith R1 (e.g., 1/T1) = 3.6F 0.3/(mmol/L)/s and R2 (1/T2) = 124F 7.2/(mmol/L)/s at 3 T (Fig. 1). Such amphiphilic polymer–coated iron oxide nanoparticles have combined characteristics ofa relatively small particle size for easy in vivo delivery, a largesurface area for conjugating biomolecules, and sufficient T2

effect for magnetic resonance imaging contrast.The amount and purity of recombinant mouse amino-

terminal fragment peptides were determined by gel electropho-resis and Western blot analysis. Coomassie blue staining of theSDS-PAGE gel revealed an amino-terminal fragment bandlocated at f17 kDa (Fig. 1). The presence of His-taggedamino-terminal fragment peptides was confirmed by Westernblot using a monoclonal anti–His tag antibody, showing astrong positive band in the location corresponding to the amino-terminal fragment-peptides identified by Coomassie blue stain-ing (Fig. 1). Conjugation of amino-terminal fragment peptides to





the negatively charged carboxyl groups on the particle sur-face was further confirmed by the reduction of surface poten-tial from an average of -30 mV to -11 mV after attachment ofamino-terminal fragment peptides, suggesting the charge neutral-ization of carboxyl groups after conjugationwith peptides (Fig. 1).From the measurement of fluorescence intensity produced fromthe Cy5.5 dye conjugated to the ATF-IO nanoparticles, weestimated that 8 to 10 amino-terminal fragment peptides wereattached to each nanoparticle (Fig. 1).Specific binding and internalization of ATF-IO nanoparticles to

uPA receptor–expressing mammary tumor cells. We examinedthe level of uPA receptor expression in human breast cancer andnormal tissues. A high level of surface uPA receptor was found inmouse mammary tumor 4T1 cells by immunofluorescencelabeling of viable cells using an anti–uPA receptor antibody.On the other hand, humanbreast cancer T47D cells that lack uPAreceptor expression were used as a negative control in this study(Fig. 2A). Consistent with previous observations, uPA receptor isstrongly expressed in invasive breast cancer tissues but is notdetected in normal breast tissues (ref. 24; Fig. 2A).

To determine whether amino-terminal fragment peptidesmaintain a high binding affinity after being conjugated to theiron oxide nanoparticles, we did a pull-down assay using celllysates obtained from uPA receptor–positive 4T1 and –negativeT47D cells. Our results showed that free amino-terminalfragment peptides and ATF-IO nanoparticles bound to andprecipitated uPA receptor proteins in the cell lysates, resulting ina positive uPA receptor band detected by Western blot assay in4T1 but not T47D cell lysates (Fig. 2B). It has been shown thatinteraction of uPA with uPA receptor leads to the internalizationof the ligand/receptor complex (35). To determine whether thebinding of amino-terminal fragment to uPA receptor leads toreceptor-mediated endocytosis, we examined 4T1 and T47D cellsafter incubation with Cy5.5–ATF-IO at 37jC for 3 hours under aconfocal microscope. We found that Cy5.5–ATF-IO nano-particles were internalized by 4T1 cells but not by T47D cells(Fig. 2C). The presence of intracellular iron oxide nanoparticlesin 4T1 cells was further shown by Prussian blue staining(Fig. 2C).Binding and internalization of ATF-IO nanoparticles in tumor

cells and magnetic resonance imaging contrast effect. Receptor-mediated binding and internalization of imaging probespromote their accumulation at the tumor site, which increasescontrast effect. Prussian blue staining detected a high level ofiron oxide nanoparticles in 4T1 cells incubated with ATF-IO butnot with nontargeted iron oxide nanoparticles. uPA receptor–negative T47D cells showed only a very low level of nonspecificuptake (Fig. 3A). 4T1 cells incubated with ATF-IO nanoparticlesshowed magnetic resonance imaging contrast with signalreduction in T2-weighted gradient echo imaging (Fig. 3B, top).T2 relaxometry measurements indicated that the T2 value of4T1 cells bound with ATF-IO nanoparticles dropped sig-nificantly compared with those of the T47D cells in-cubated with ATF-IO oxide nanoparticles and the samplestreated with nontargeted iron oxide nanoparticles (Fig. 3B,bottom). Because the T2 value is a function of the iron con-centration, the T2 relaxometry data suggest that reduction ofT2 relaxation time and magnetic resonance imaging signalin T2 weighted imaging are induced by the specific bindingof ATF-IO nanoparticles to the uPA receptor–expressing4T1 cells.

In vivo targeting and magnetic resonance imaging of s.c.mammary tumors in mice. In BALB/c mice bearing s.c. mousemammary tumors derived from the 4T1 tumor cell line,T2-weighted gradient echo imaging and multi-echo fast spin

Fig. 2. Specificityofamino-terminal fragmentpeptidesorATF-IOnanoparticles.A, immunofluorescence labeling.Ahigh levelofurokinaseplasminogenactivatorreceptor (red) is detected in frozen tissue sectionsof invasivehumanbreast cancertissuesbutnot innormalbreast tissueobtained from the samepatient (top).Blue,Hoechst 33342background staining.Surface labelingof viable cells usinganti ^ urokinaseplasminogenactivator receptorantibody shows ahigh levelofurokinaseplasminogenactivator receptor in 4T1cells butnot inT47Dcells(green,bottom).B, immunoprecipitationassay. Lane1, free amino-terminalfragment (ATF)peptidespulleddownurokinaseplasminogenactivator receptor(uPAR)protein from4T1cell lysates; lane2,ATF-IOnanoparticles are alsocapable ofbindingandpulling (uPAR)protein; lane3, control total lysatesof 4T1cells; lanes4 and5, theuPARproteinbandis notdetectedafterincubationof freeATFpeptides (lane4) orATF-IOnanoparticles (lane5)withT47Dcell lysates.C, specificbindingandinternalizationofCy5.5^ ATF-IOnanoparticles inurokinaseplasminogenactivator receptor ^ expressingtumorcells.Confocal images show intracellular localizationofCy5.5fluorescence(red) in4T1butnot inT47Dcells (top). Prussianblue stainingfurther confirms thepresenceofblue iron staining inside the4T1cells (bottom).





echo imaging showed that ATF-IO nanoparticles selectivelyaccumulated in tumors, as evidenced by a reduction in T2 valuesand signal decrease in T2-weighted images in various areas of thetumor mass (Fig. 4A). The region of interest analysis of magneticresonance imaging signal change showed a 3-fold signalreduction in animals receiving ATF-IO nanoparticles whencompared with that in mice receiving nontargeted iron oxideparticles (Fig. 4B). Although we observed decreases in magnetic

resonance imaging signals in the liver and spleen in the miceinjected with ATF-IO nanoparticles because of an iron oxideparticle–induced T2 effect, the reduction in magnetic resonanceimaging signal was 50% (liver) to 80% (spleen) less than that inmice that received nontargeted iron oxide nanoparticles,suggesting that liver and spleen uptake of the nanoparticleswas reduced for ATF-IO nanoparticles (Fig. 4B). To furtherconfirm the distribution of nontargeted and ATF-IO nano-particles in normal and tumor tissues, Prussian blue staining wasdone on tissue sections obtained from the mice that receivedcontrol iron oxide or ATF-IO nanoparticles. Prussian blue–stained cells were detected in the tumor sections of animals thatreceived ATF-IO nanoparticles but not in sections from animalsthat received nontargeted iron oxide nanoparticles. Highmagnification images showed the intracellular localization ofiron oxide nanoparticles in the cells (Fig. 4C). In normal tissues,we found high levels of Prussian blue–positive cells in the liverand spleen of the mice that received nontargeted iron oxidenanoparticles. However, liver and spleen tissue sections from thegroup receiving ATF-IO nanoparticles had fewer positive cells.We did not detect iron oxide nanoparticles in the tissue sectionsof the brain or heart from mice injected with either nontargetediron oxide or ATF-IO nanoparticles (Fig. 4C). For both groups,the lung and kidney tissues were also negative in most cases(Fig. 4C) and only a few scattered iron-positive cells weredetected in some sections.Targeted magnetic resonance imaging of i.p. mammary tumor

lesions using ATF-IO nanoparticles. We tested the feasibility oftargeting and in vivo imaging of metastatic lesions using ananimal model bearing i.p. 4T1 tumors. The presence anddevelopment of the 4T1 tumor was determined and followedby bioluminescence imaging (36). At 5 hours after injection ofthe ATF-IO nanoparticles, magnetic resonance imaging signalsdecreased in two tumor lesions located on top of the right kidney(Fig. 5, top). Themagnetic resonance imaging signal in the regionrecovered gradually 30 hours after the injection of the ATF-IOnanoparticles. In contrast, we did not detect an iron oxidenanoparticle-inducedmagnetic resonance imaging signal changein a tumor-bearing mouse after injection of nontargeted ironoxide (Fig. 5, bottom). The selective accumulation of uPAreceptor–targeted ATF-IO nanoparticles in this metastatic i.p.tumor model was further confirmed by Prussian blue staining oftissue sections from the sacrificed animals. A high percentage ofiron-positive cells was found in the tumor lesion, whereas thekidney beneath the tumorwas negative (Fig. 5, top). On the otherhand, we did not detect iron-positive cells in tissue sections of thei.p. tumor mass obtained from the mouse that receivednontargeted iron oxide nanoparticles, whereas the adjacentnormal liver tissue had a high level of iron-positive cells (Fig. 5,bottom).Validation of tumor-targeted magnetic resonance imaging in 4T1

mouse mammary tumors using near-IR optical imaging. Near-IRoptical imaging provides a simple and sensitive approachto confirm tumor targeting and to monitor the distributionof Cy5.5–ATF-IO nanoparticles in small animals. When moni-toring mice bearing s.c. 4T1 tumors at different time points afterinjection of Cy5.5–ATF-IO nanoparticles, we observed a strongnear-IR signal in the tumormass 24 hours after administration ofthe nanoparticles. The signal intensity in the tumor massincreased to a peak level at 48 hours but declined at 72 hours(Fig. 6A). We also detected near-IR signals in kidneys,

Fig. 3. ATF-IOnanoparticle ^ inducedmagnetic resonance imagingsignal changeafterbinding to tumorcells.A, Prussianblue staining shows specificbindingandinternalizationofATF-IOnanoparticles in4T1cells.A lowlevelof scatteredbluestaining is foundin4T1cells incubatedwithnontargetedironoxidenanoparticles orurokinaseplasminogenactivator receptor ^ negativeT47Dcells incubatedwitheither ironoxide (IO)orATF-IOnanoparticles.B, reductionofT2 values in4T1cells(top) butnot inT47Dcellsmeasuredbymagnetic resonance imagingT2 relaxometryimplies the specificbindingofATF-IOnanoparticles tourokinaseplasminogenactivator receptor ^ expressingcells.A lowT2 value (orange-to-redcolor) correlateswithahighironconcentration.The fastestT2 value dropwasdetected in4T1cellsincubatedwithATF-IOnanoparticles (bottom, redcurve). Scalebar, echo time(TE)inmilliseconds.T2 valuesofeach sample/wellwere calculated frommulti-echoimagesby fitting thedecaycurveonapixel-by-pixelbasisusing thenonlinearmono-exponential algorithmofMi =M0 * exp (-TEi/T2).Mi is the intensityormagnitudeof themeasurement at a timepointof i.M0 is the intensityormagnitudeof themeasurement at the timeof zero.





suggesting that some Cy5.5–ATF-IO nanoparticle and/orCy5.5–amino-terminal fragment dissociated from iron oxidenanoparticles may be eliminated through the kidney. Fur-thermore, we found that areas with magnetic resonanceimaging contrast change in the tumor overlapped well withthe presence of near-IR signal when comparing optical andmagnetic resonance images (Fig. 6B). Examination of thetissue sections with positive Prussian blue staining revealedthat Cy5.5 near-IR signals colocalized with blue iron-positivecells (Fig. 6C).Reduced nonspecific uptake of amino-terminal fragment–iron

oxide nanoparticles. Previous studies reported that iron oxidenanoparticles can be nonspecifically taken up by macrophages,as well as the reticuloendothelial system in the liver and spleen.We examined the tumor tissue sections stained with bothPrussian blue and an antibody to CD68, a marker for macro-

phages (37). We found iron-positive cells present in CD68-positive and -negative cell populations. A high percentage ofiron-positive cells did not express CD68, suggesting thatnonmacrophage cell types such as tumor cells contain ironoxide nanoparticles. It has been shown that active macrophagesin breast cancer tissues also express a high level of uPA receptor(21, 38). It is possible that the presence of iron-positivemacrophages may be due to active targeting of uPA receptor–expressing macrophages rather than nonspecific uptake of ironoxide nanoparticles.

Additionally, a magnetic resonance imaging T2 map of amammary tumor showed that accumulation of amino-terminalfragment–iron oxide nanoparticles was not uniformly distrib-uted inside the tumor mass. At various sections of tumormagnetic resonance images, magnetic resonance imaging signaldecreases were heterogeneous in the tumor, suggesting that

Fig. 4. Invivomagnetic resonance imagingof 4T1mammary tumorusingATF-IOnanoparticles.A,magnetic resonance imagingofs.c.mammary tumor.Amarkedmagneticresonance imaging signaldropwithT2weightedcontrastwasobservedin s.c. tumorareas (pinkdashed lined) 6hafter the tail veinATF-IOnanoparticles.Heterogeneous signal changessuggest that intratumoraldistributionofATF-IOnanoparticles isnotuniformin the s.c. tumor(redarrow).T2 contrast change is also foundinthe liver (yellowarrows andpinkasterisk).Selectedmagnetic resonance image is arepresentative imageof sevenmice thatreceivedATF-IOnanoparticles.Amagneticresonance imageof themouse that receivednontargeted ironoxidenanoparticles is inFig. 6B.B, organ-specificprofilingofmagnetic resonanceimagingsignalchange.Signalchanges inthemicethat receivednontargeted ironoxideorATF-IOnanoparticles for6hweremeasuredin theregionsof tumoror variousnormal tissues.Relative intensitywas calculatedusing theintensity in the legmuscle as a reference. Folddecreases in the intensityof themagneticresonance imagewere comparedbetweenpre ^ andpost ^ ATF-IOnanoparticle injectionsandplotted in the figure.Barplot,meanvaluesof three regions.C, Prussianblue stainingof theironoxidenanoparticles in tumorandnormal tissues48hafter injectionof thenanoparticles.Red, background stainingwithnuclear fast red.





intratumoral distribution of the targeted nanoparticles varies indifferent areas (Fig. 6C). Interestingly, areas with the greatestdecline in signal were largely in the periphery regions of thetumor mass, which are rich in blood vessels relative to thenecrotic areas at the center of the tumor.

Discussion

Molecular imaging probes targeting specific cancer markershave been long sought after in applying tumor imaging fordisease-specific detection and personalized therapeutics. How-ever, the development of receptor-targeted imaging and its in vivoapplications are particularly challenging, with current obstaclesincluding (a) the identification of cell-surface biomarkers thatare expressed sufficiently in tumor cells or tumor environmentsfor sensitive tumor imaging, (b) the production of stable and

high-affinity targeting ligands in large amounts for in vivostudies, and (c) the development of safe and biodegradablecontrast agents that can also produce strong imaging signal orcontrast.

The uPA receptor–targeted iron oxide nanoparticle probereported here provides an example that addresses thesechallenges and shows the feasibility of in vivo receptor-targetedtumor imaging with MRI at clinical field strength. Results of ourstudy showed that amino-terminal fragment–iron oxide nano-particles are capable of targeting uPA receptor–expressing tumorcells in vitro and in vivo and enable receptor-targeted magneticresonance and optical imaging of the tumor in vivo. We believethat the following characteristics of amino-terminal fragment–iron oxide nanoparticles enabled uPA receptor–targeted imag-ing in vivo. First, we used a tumor-targeting ligand from a naturalhigh-affinity receptor-binding domain of uPA. uPA is composed

Fig. 5. Targetingand invivomagnetic resonance tumor imagingof i.p.mammary tumor lesions.Top, bioluminescence imagingdetects thepresenceof i.p. tumorson theupperrightof theperitonealcavityof themouse.Magnetic resonance images reveal twoareas locatednear the right kidney(reddashed lined)withdecreasedmagnetic resonanceimagingsignals 5or30hafter the tail vein injectionof11.2nmol/kgofbodyweightofATF-IOnanoparticles,whichcorresponded to the locations detectedbybioluminescenceimaging. Examinationof themouseperitonealcavityconfirmed the locationsof two tumor lesionson the topof the right kidney.Specific accumulationofATF-IOnanoparticles inthe tumor tissueswasconfirmedbyPrussianbluestaining.Bottom, thecontrolmousewithatumorlesionintheupper rightof theperitonealcavity receivednontargetedironoxide(IO)nanoparticles.There isnomagnetic resonance imagingcontrast change in the tumorarea (pinkdashed lined) 6hafteradministrationof ironoxidenanoparticles,whereasthe liverof themouse shows amarkedcontrastdecrease. Prussianblue stainingof tissue sections revealed iron-positive cells in thenormal livernear the tumor regionbutnot inthe tumor tissues (A reddashed line dividesnormal liverand tumor tissue areas).

Fig. 6. Dualmodality imagingof s.c.4T1mousemammary tumorusingCy5.5^ ATF-IOnanoparticles.A, near-IRoptical imagingof tumor targetingand tissue distributionofCy5.5^ ATF-IOnanoparticlesover time.Anudemousebearingans.c.4T1tumorreceived200pmolpermouseofCy5.5^ ATF-IOnanoparticlesin50ALPBS,whichwascalculatedfromthe ironoxidenanoparticle concentration.Near-IRoptical imagingwasdoneatdifferent timepoints. Pinkarrows, s.c. tumor; greenarrows, kidneys.Backand side imagesare at 72h.B, simultaneousmagnetic resonanceandoptical imagingofmammary tumor.A tumor-bearingmouse receivedCy5.5^ ATF-IOnanoparticles showeda signaldropafter 48hin the lower-left halfof the s.c. tumor inT2-weightedmagnetic resonance image.Optical imaging reveals thenear-IR signal in the tumorareacorrespondingwellwith themagnetic resonance imaging results (bottom,pinkdashed lined).T2 contrast change isnotdetectedin the s.c. tumorof themouse that receivednontargetedironoxidenanoparticles (top).Magnetic resonance image is the results ofa totalof sevencontrol tumor-bearingmice that receivedironoxidenanoparticles.C, examinationof thelocationofATF-IOnanoparticles andCy5.5 signal in tumor tissues.Double labelingofPrussianblue stainingandanAlexaFluor488^ anti-CD68antibody reveals thatmanyblue iron-positive cells arenegative forCD68(redarrow).Moreover, colocalizationof ironstainingandCy5.5 signal is also detectedin the tumor tissues (white arrows).D,T2mapofa series ofmagnetic resonance images taken fromans.c.mammary tumor lesionusingmulti-echoT2-weighted spinechomagnetic resonance imaging.Top, controlmagnetic resonance images takenbeforeATF-IOnanoparticle injection.Bottom, correspondingmagnetic resonance images taken48hafter injectionofATF-IOnanoparticles.Orange-to-redcoloredareas, tumorareaswith the greatest reductionofT2 relaxation time.Regionalof interest analysis ofT2mappingwasused.T2map typicallywasobtained for thewholemouse, butonly the tumoras regionof interestwasdisplayedhere topresent anoverlayofT2 valueswithanatomic images.





of three independently folded domain structures: growth factordomain, Kringle domain, and serine protease domain. uPAbindswith high affinity to uPA receptor through the amino-terminalfragment of the growth factor domain, with a dissociationconstant (Kd) < 1 nmol/L (39). Studies have shown that amino-terminal fragment (residues 1-135 amino acid) of uPA is a potentantagonist of uPA/uPA receptor binding because amino-terminalfragment lacks the serine protease domain of uPA, whichnegatively regulates its function by cleaving uPA receptor into anonbinding receptor (40, 41). Second, efficient internalizationof the ligand/receptor complex may increase the concentrationof the iron oxide nanoparticles in tumor cells, which enhancesthe effect of uPA receptor–targeted tumor imaging (35, 42).Third, nanoparticles provide favorable pharmacokinetics byprolonging blood circulation time, allowing sufficient amountsof nanoparticles to reach the tumor. Furthermore, our ability toproduce the recombinant protein in a large scale is essential forpreclinical and eventually clinical studies. It should be men-tioned that this study was done using mouse amino-terminalfragment peptides and the 4T1 mouse tumor model toinvestigate the feasibility of targeting uPA receptor. Although ithas been shown that the interaction of uPA with its receptor hasspecies specificity (43), we found that mouse amino-terminalfragment peptides bind efficiently to mouse tumor cells and alsoshow cross-reactivity with human uPA receptor–expressingtumor cells. A major advantage of using mouse amino-terminalfragment peptides to conduct studies in a mouse tumor model isthat the targeting specificity and biodistribution in normaltissues of this imaging probe can be studied in greater detail. Forfuture clinical application, it is desirable to use a human amino-terminal fragment peptide. We have produced recombinanthuman amino-terminal fragment peptides and have showntarget specificity in human breast cancer xenograft models innude mice.7

Extensive studies have shown that human breast cancer andtumor stromal cells have a much higher level of uPA receptorcompared with normal breast tissues (44, 45). Differencesbetween the level of uPA receptor present on the surface ofnormal and tumor cells suggest that an elevated uPA receptor incancer may be sufficient for in vivo uPA receptor–targeted tumorimaging. Several studies have shown that the highest level of uPAreceptor expression is detected in the invasive edge of the tumorregions (23, 28), which are usually enriched in blood vessels,making this area particularly accessible for uPA receptor–targeted iron oxide nanoparticles. In addition, the ability ofnanoparticles to leak across tumor endothelium but not normalvasculatures by passive targeting may prevent the interaction oftargeted nanoparticles with some cell types in normal tissues thatexpress a low level of uPA receptor.

In this study,wepreparedhigh-quality and uniformly sized ironoxide nanoparticles with a thin ampiphilic copolymer coating(estimated at 2 nm). Compared with conventional dextran orpolyethylene glycol-coated nanoparticles used previously, ampi-philic copolymer–coated iron oxide nanoparticles form arelatively small particle complex (f18 nm in this study), whichis desirable for in vivo delivery of the imaging probe. Severalprevious studies used a commercially available superparamagneticiron oxide nanoparticle, Feridex, which has a particle core size

distribution of 20 to 30 nm or 80 to 150 nm in overall diameterwith dextran coating (13). We believe that size uniformity isessential for potential quantification using magnetic resonanceimaging signal of the amount of probe in vivo.

Although other small molecule imaging agents may have betterintratumoral distribution than nanoparticle-based imagingagents, these imaging agents are usually eliminated from theblood circulation in a relatively short time (<30 minutes), whichmakes it unlikely that sufficient levels of the targeted contrastagents can accumulate at the tumor site (2). It has been shown thatpolymer-coated iron oxide nanoparticles have >8 hours of plasmaretention time (11). This longer circulation time could be animportant factor enabling targeted iron oxide nanoparticles toreach the tumor site and bind to tumor cells. amino-terminalfragment–iron oxide nanoparticles were stable in vivo and inintracellular environments for >48 hours during our imagingexperiments. We observed that the intratumoral near-IR signalincreased over time and reached its highest level around 48 hoursafter the administration of iron oxide nanoparticles, suggestingthat the long blood retention time may facilitate nanoparticletumor targeting.

It has been reported by several other groups that largeproportions of magnetic iron oxide nanoparticles are taken upby the reticuloendothelial system in the liver and spleen and thenare subsequently metabolized or used for iron storage (12, 13).Our data showed that amino-terminal fragment–iron oxidenanoparticles have reduced liver and spleen uptake comparedwith nontargeted iron oxide nanoparticles. This suggests thatconjugation of amino-terminal fragment-peptides to the ironoxide nanoparticles attenuates their nonspecific capture andretention in the liver and spleen, which commonly occurs aftersystemic delivery.

In conclusion, we have developed a uPA receptor–targetedmolecular imagingnanoprobe that has a uniform-sized ironoxidenanocrystal core, a thin ampiphilic copolymer coating, and a highaffinity receptor binding domain of uPA conjugatedwith a near-IRdye. This receptor-targeted nanoprobe selectively binds to and isinternalized by tumor cells and can specifically accumulate inprimary and metastatic tumors, facilitating in vivo magneticresonance and optical imaging in a mouse mammary tumormodel. Such uPA receptor–targeted imaging nanoparticles arepromising probes for molecular magnetic resonance imaging ofbreast cancer and several other cancer types, such as pancreatic,lung, and brain, which express high levels of uPA receptor (46–48). Given that chemotherapy drugs can be incorporated into thetargetednanoparticles (49), it will be feasible touse uPA receptor–targeted iron oxide nanoparticles for delivery of therapeutic agentsinto tumor cells and monitoring the response to therapy usingmagnetic resonance imaging.

Disclosure of Potential Conflicts of Interest

Nopotential conflicts of interestwere disclosed.

Acknowledgments

We thankDr. AntheaHammond for her critical editing of themanuscript, Dr.MarkW. Dewhirst for kindly providing us with luciferase gene ^ stable 4T1cell line, Drs.AdamMarcus and Katherine Schafer-Hales in the Cell Imaging Core of theWinshipCancer Institute for their assistance in confocal microscopy, and Dr. Hongwei Duanforhishelpwith zetapotential andparticle size analysis.

7 H. Sajja, Z. Cao, and L.Yang, unpublished observation.







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2009;15:4722-4732. Clin Cancer Res Lily Yang, Xiang-Hong Peng, Y. Andrew Wang, et al. Breast Cancer

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