齐墩果酸药理活性

Hybrid PET/MR imaging of tumors using an oleanolic acid-conjugated nanoparticle

Sung-min Kim1,Min Kyung Chae1,Min Su Yim,Il Ha Jeong,Janggeun Cho,

Chulhyun Lee**,Eun Kyoung Ryu*

Center of Magnetic Resonance Research,Korea Basic Science Institute(KBSI),Chungbuk363-883,Republic of Korea

a r t i c l e i n f o

Article history:

Received27June2013 Accepted22July2013 Available online8August2013

Keywords:

Oleanolic acid

Iron oxide nanoparticle

PET/MRI

Diagnosis

Colon cancer a b s t r a c t

Research into multifunctional nanoparticles is focused on creating an agent for use in an all-in-one multimodal imaging system that includes diagnostic imaging,drug delivery,and therapeutic moni-toring.We designed a new dual-modality tumor-targeting agent with a new tumor-targeting molecule, oleanolic acid(OA),which is derived from a natural compound and coupled with a macrocyclic chelating agent such as1,4,7-triazacyclononane-1,4,7-triacetic acid(NOTA),iron oxide nanoparticles(IONP),and radiolabeling components such as68Ga for dual-modality positron emission tomography(PET)/magnetic resonance imaging(MRI).We attempted to obtain fusion PET/MR images with the68Ga e NOTA e OA e IONP hybrid tumor-targeting imaging agent using colon cancer(HT-29)xenograft mice models.The HT-29cancer cells showed high uptake of68Ga e NOTA e OA e IONP,which also had an inhibitory effect on the cells.Moreover,we obtained PET and MRI tumor images as well as fusion PET/MRI images of the tumors using68Ga e NOTA e OA e IONP.Therefore,the dual-modality cancer-targeting radiolabeled nanoparticle reported here is a potent imaging agent that is suitable for PET,MRI,and PET/MRI-based diagnosis of tumors;it also has the advantage of not only detecting tumor functionality,but also simultaneously aiding in tumor resolution.

Crown Copyrightó2013Published by Elsevier Ltd.All rights reserved.

1.Introduction

Early detection of disease is a popular and important issue in current medical practice.Several imaging modalities are presently used for the diagnosis of human diseases,but despite the advanced technology used in such diagnostic instruments,it has become increasingly clear that a multimodal imaging modality may be crucial for precise detection of cancer.The combination of different imaging modalities overcomes the inherent disadvantages of each single instrument.Positron emission tomography(PET)has high signal sensitivity,but it does not provide any information regarding anatomical structure,which thus requires the use of single photon emission computerized tomography(CT).CT is useful for obtaining clear in vivo images of anatomical structures,and is especially economically favorable compared with other imaging modalities, but it is not useful for soft tissue imaging as it yields poor-quality images of soft tissues[1,2].Magnetic resonance imaging(MRI)provides excellent spatial resolution without any ionizing radiation, but it has low sensitivity.Consequently,the combination of PET and MRI should allow a more accurate diagnosis by overcoming the limitations particular to each technique without exposing the subject to ionizing radiation;it can also be helpful for improving our understanding of certain malignant and/or benign cancers in soft tissues[3e5].

Recently,multifunctional nanoparticles have been designed for use in multimodal imaging systems that combine diagnostic im-aging,drug delivery,and therapeutic monitoring[6,7].One example is a combination of an anti-cancer drug delivery/PET/MR imaging agent and a PET/MRI/?uorescence imaging agent.Nano-particles have a large surface area and functional groups can easily be introduced by appropriate surface modi?cation.In addition, nanoparticles have a long circulation time in the body due to their size(100nm)and they accumulate in lesions,which makes them ef?cient agents for drug delivery[8e11].

To date,many different types of multimodal imaging agents have been reported[12e15].Usually,these are composed of iron oxide nanoparticles(IONP),tumor-targeting molecules,and radioactive labeling components.Iron oxide nanoparticles are used as MRI contrast agents,and have been coated with substances

*Corresponding author.Tel.:t82432405091;fax:t82432405419.

**Corresponding author.Tel.:t82432405090;fax:t82432405419.

E-mail addresses:chulhyun@kbsi.re.kr(C.Lee),ekryu@kbsi.re.kr(E.K.Ryu). 1These authors contributed equally to this

work.Contents lists available at ScienceDirect Biomaterials

journal h omepage:

https://www.360docs.net/doc/2f18304816.html,/locate/biomaterials

0142-9612/$e see front matter Crown Copyrightó2013Published by Elsevier Ltd.All rights reserved. https://www.360docs.net/doc/2f18304816.html,/10.1016/j.biomaterials.2013.07.078

Biomaterials34(2013)8114e8121

such as dopamine,dextran,protein,and a functionalized poly-ethyleneglycol(PEG)-based polymer due to their stealth effect. These nanoparticles are stable in biological environments and are protected from unwanted non-speci?c binding and reticuloendo-thelial system uptake(RES)[16e18].

For multimodal imaging,functionalized IONP are coupled with macrocyclic chelating agents such as1,4,7-triazacyclononane-1,4,7-triacetic acid(NOTA)and1,4,7,10-tetraazacyclododecane-N-N0,N00,N000,-tetraacetic acid(DOTA)and are used for PET imaging and with tumor-targeting ligands(i.e.,cyclo(Arg e Gly e Asp e D e Phe e Cys)[c(RGDfC)],doxorubicin,and antibodies).The RGD class is the most promising of the tumor-targeting ligands.For example, Chen et al.synthesized an RGD-conjugated radiolabeled IONP and successfully obtained dual PET/MRI images by targeting a v b3 integrin expression by a tumor[12].An anti-tumor drug delivery and PET/MRI dual-imaging agent comprising radiolabeled IONP, doxorubicin(anti-tumor agent),and cRGD have also been reported [14].Target-speci?c binding is an important concept as it improves our understanding of the progression of disease and of drug effects using molecular imaging.

Several criteria have to be considered when developing imaging agents for human applications,including their pharmacokinetics, solubility,lipophilicity,ef?cacy,and toxicity.We consider toxicity and speci?c and selective targeting of a tumor to be the most important of these criteria.Oleanolic acid(3b-hydroxy-olea-12-en-28-oic acid,OA)is a non-toxic natural compound that is a member of the triterpenoid family.It is known to have potent hepatoprotective, antibacterial,antifungal,and anti-in?ammatory properties[19,20]. Moreover,OA has been shown to inhibit cancer cell proliferation and to induce apoptosis in cancer cells[21,22].In our previous studies,we found that OA derivatives showed strong inhibition of cancer cells, especially colon cancer cells,and induced apoptosis and cancer cell death.We have since attempted to develop an imaging agent for PET that combines both high tumor uptake and tumor-speci?c targeting.

In the present study,we report our design of a dual-modality tumor-targeting agent comprising a new tumor-targeting molecule (OA),IONP,and a radioactive chelator(NOTA)for PET/MRI dual im-aging.We synthesized IONP by a thermal decomposition reaction and by changing the coating material to a DSPE-mPEG2000/DSPE-mPEG2000amine,which made it possible for the agent to be dispersed in water.In addition,the functional amine groups of the PEG e phospholipid made it possible to introduce a tumor-targeting and radioactive ion chelating ligand for use as an MRI contrast agent.NOTA was used as the radioactive chelating ligand in order to bind radioactive isotopes(e.g.,68Ga,t1/2?68min)for PET.Finally,we attempted to obtain fusion PET/MR images with this hybrid tumor-targeting imaging agent using xenograft mice models of colon cancer.

2.Materials and methods

2.1.Preparation of IONP e OA e NOTA

2.1.1.Chemicals

All reagents were used without further puri?cation.Oleanolic acid,iron(III) acetylacetonate([Fe(acac)3])(99.9%),oleylamine(>70%),and diisopropylethylamine (DIEA)were purchased from Aldrich Chemical Co.(St.Louis,MO,USA).Oleic acid (90%)was purchased from Alfa-Aesar(Ward Hill,MA,USA).1,2-Distearoyl-sn-glyc-ero-3-phosphoethanol-amine-N-methoxy(polyethylene-glycol)2000(DSPE-mPEG 2000)and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino-(polyethyl-englycol)2000](DSPE-mPEG2000amine)were purchased from Avanti Polar Lipids, Inc.(Huntsville,Ala,USA).O-Benzotriazole-N,N,N,N0-tetramethyl-uronium-hexa-?uoro-phosphate(HBTU)and hydroxybenzotriazole(HOBt)were purchased from BeadTech Inc.(Seoul,Korea),and(p-SCN-Bn)-NOTA$3HCl(NOTA)was procured from FutureChem Co.,Ltd.(Seoul,Korea).

2.1.2.Synthesis of Fe3O4nanoparticles

Fe3O4nanoparticles were synthesized by a reductive thermal decomposition reaction.Fe(acac)3(1.42g,4.02mmol),oleic acid(4mL,12.6mmol),oleylamine (4mL,12.4mmol),and1,4-hexadecanediol(5.15g,20mmol)were mixed and stirred at120 C with vigorous stirring for2h and then partially vacuumed to simulta-neously remove moisture and oxygen.The solution was then heated to200 C under argon and kept at this temperature for2h.Thereafter,the solution temperature was rapidly increased to300 C and kept at this temperature for30min.The solution turned a dark-brown color and was cooled down to room temperature and washed with ethanol.The nanoparticles were redispersed into hexane and precipitated by the addition of excess ethanol and then puri?ed by centrifugation.The?nal products were dispersed into hexane and stored under an argon atmosphere.

2.1.

3.Coating of Fe3O4nanoparticles(IONP)with PEG e phospholipid

A total of1mL of Fe3O4nanoparticles(3.3mg)in CHCl3was mixed with1mL of CHCl3containing76.5mg(4:1ratio of DSPE-mPEG2000:DSPE-mPEG2000amine) of DSPE-mPEG2000and DSPE-mPEG2000amine.The reaction mixture was then mixed for1h at room temperature.Thereafter,the mixture was dried under argon gas and left in a vacuum oven at40 C for1h to remove all traces of the organic solvent.

2.1.4.Coupling of the nanoparticles and oleanolic acid(OA e IONP)

The IONP?lm was dispersed in DMF(1mL)and mixed with OA(2mg),O-ben-zotriazole-N,N,N,N0-tetramethyl-uronium-hexa?uoro-phosphate(HBTU,4equiv.) and hydroxybenzotriazole(HOBt,4equiv.).The solution was then incubated in the basic condition(diisopropylethylamine,DIEA)at room temperature overnight.Next, the reaction mixture was washed with distilled water using a100kDa ultra?ltration unit,the Amicon Ultra-4(Millipore Corp.).The?nal OA e IONP complex was stored in 10m M borate buffer at4 C.

2.1.5.Coupling of OA e nanoparticles and NOTA(NOTA e OA e IONP)

The OA e IONP solution was buffer-changed with0.1M Na2CO3using a100kDa ultra?ltration unit.NOTA(3.6mg)was added into the OA e IONP solution and mixed for2h.The reaction mixture was then washed in distilled water by using a100kDa ultra?ltration unit,and?nally puri?ed through a PD-10column with10m M borate buffer.

2.2.Radiolabeling of NOTA e OA e nanoparticles(68Ga e NOTA e OA e IONP)

The radioisotope,68Ga,was produced with a68Ge/68Ga generator(Eckert& Ziegler,Berlin,Germany)using0.1N HCl solution.For68Ga labeling of the nano-particles(68Ga e NOTAOA e IONP),NOTA e OA e IONP(5.8m mol)was dissolved in 300m L of0.5M phosphate buffer(pH7.4)added to the68Ga/0.1N HCl solution.The mixture was then adjusted to a pH of5.0e5.5,stirred for20min to induce a reaction at room temperature,and then puri?ed using size exclusion chromatography(PD-10 column).

2.3.Characterization of the synthesized nanoparticles

2.3.1.Measurement of the nanoparticles

A sample for transmission electron microscopy(TEM)analysis was prepared by drying a dispersion of the synthesized nanoparticles in solvent on amorphous carbon-coated copper grids.The particles were imaged using an FEI Tecnai G2F30ST microscope.The size distribution of the nanoparticles dispersed in solvent was characterized by dynamic light scattering(DLS)using a Nanosizer(Zetasizer Nano ZS,Malvern,U.K.).

2.3.2.Veri?cation of NOTA e OA e IONP by FT-IR spectroscopy

The FT-IR spectra of the nanoparticles were obtained with a microscopic FT-IR/ Raman Spectrometer(Vertex80V,Bruker,Germany).The powder samples(OA and NOTA e OA e IONP)were transformed into a thin plate with KBr pellet.

2.3.3.Measurement of T2relaxation properties

All MRI experiments were performed with a4.7T MRI(Bruker Biospec,Ger-many)equipped with a72-mm inner diameter quadrature RF coil at the Korea Basic Science Institute in Ochang,and elemental analysis of iron in the hydrophilic nanoparticle samples was performed using an inductive coupled plasma-atomic emission spectrometer(ICP-AES;Optima4300DV;PerkinElmer,USA)at the Korea Basic Science Institute in Gwangju.T2relaxation maps of OA e IONP and NOTA e OA e IONP were measured using the MSME sequence(TR/TE?1000/8ms,?ip angle?180)with a FOV of5?5cm and a slice thickness of2mm.The r2values were calculated by?tting a curve to1/T2relaxation time(sà1)versus the iron ion con-centration(m M).

2.4.Proliferation of nanoparticles in colon cancer

2.4.1.Cancer cell line

Colon cancer(HT-29)cells were obtained from a human cell line(KCLBò).The cells were cultured in RPMI1640(Gibco,Grand Island,New York,USA)supple-mented with10%heat-inactivated fetal bovine serum(FBS,Gibco)and penicillin (100units/mL).The culture was maintained at37 C in5%CO2.

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2.4.2.Tumor cell proliferation determined by XTT assay

The toxicity of the nanoparticles was determined using a sodium 3-[1-(phe-nylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate (XTT)solution (Cell Proliferation Kit II,Roche Diagnostics,Mannheim,Germany).The cells (1?103cells/well)were cultured on 96-well microplates (NunC,Denmark)containing RPMI-1640medium with 10%FBS for 24h,and then exposed to various concentrations (0,0.5,1,2,4,8,16,32,64,128,256,and 512m M )of IONP or OA-conjugated IONP and incubated for a further 24h at 37 C under 5%CO 2.Thereafter,10m L of XTT solution (1mg/mL)was mixed in 90m L of RPMI-1640me-dium and added to each well.Then,the proliferation rates of the cells were assessed with an ELISA reader (Molecular Devices,CA)at 490nm.The average of 4wells was used for analysis (mean ?SD).Control cells were treated with 0.1%DMSO alone.2.4.3.Cellular uptake of nanoparticles

Cellular uptake and binding with cell lysates of nanoparticles were visualized with Perls ’Prussian blue staining.HT-29cancer cells (2?104cells/well)were grown in 24-well polystyrene plates containing RPMI-1640with 10%FBS for 24h.The cells were then incubated for 3h with 30m M of nanoparticles and then ?xed with 4%paraformaldehyde.They were subsequently stained with potassium hex-acyanoferrate solution (4%potassium ferrocyanide/6%HCl,1:1v /v ,Sigma,St.Louis,MO,USA)for 30min.We used cancer cell lysates to assess binding with speci ?c cancer cell proteins.Whole cell lysates were separated by 10%SDS-PAGE (SDS-polyacrylamide gel electrophoresis),and transferred to a polyvinylidene di ?uoride (PVDF)membrane.The PVDF membrane was then incubated with 30m M of IONP or OA e IONP for 1h at room temperature,and stained with Perls ’Prussian blue staining solution.The stained membranes were observed under an inverted optical micro-scope (OLYMPUS IX81,Olympus Inc.,Japan)after counter-staining with 0.02%neutral red (Sigma,St.Louis,MO,USA).

2.5.In vitro binding of radiolabeled nanoparticles

Human colon cancer cells (HT-29)were cultured in RPMI 1640with 10%FBS and 50mg/mL penicillin/streptomycin.The cells were maintained at 37 C in 5%CO 2in air.They were seeded in 24-well plates at a density of 1?106cells/well.Their in vitro binding af ?nity was assessed by incubating the cells for 1,2,and 4h with 68Ga e NOTA e IONP or 68Ga e NOTA e OA e IONP.The cells were then added to 0.5mL of 0.1N NaOH and collected,and their radioactivity was determined using a g -counter (Beckman Coulter Inc.,Fullerton,CA,USA).2.6.In vivo hybrid PET/MRI tumor imaging

2.6.1.Animals

Animal experiments were performed according to a protocol approved by the local Institutional Review Committee on Animal Care (KBSI-AEC1001).BALB/c nu/nu mice (age 5weeks,male)were purchased from Central Lab.Animal Inc.(Seoul,Korea),and maintained under speci ?c pathogen-free conditions.Xenograft tumors were induced by subcutaneous injection of HT-29cells (a density of 1?106cells)into the dorsal region of the right thigh of each mouse.

2.6.2.MR imaging

Using a standard fast spin echo T2-weighted pulse sequence,15slices were acquired for each mouse.The acquisition parameters were as follows:TR/TE ?3500/36ms,FOV ?50.0?50.0mm,matrix size ?256?256mm,and slice thickness ?1.0mm.Parameters were monitored using a time series of MR images (from 0min to 105min).All MRI data measurements were made using a 4.7T animal MRI scanner (BioSpec 47/40;Bruker,Germany)with a 35mm volume coil.For im-aging,1mg/mL of IONP or OA e IONP was injected into the tail vein of a tumor-bearing mouse at 1,2,and 4h.The signal intensities were evaluated by the region of interest (ROI)method.The ROI included liver tissue with no obviously large vessels and the tumor.The relative signal intensities of the liver and tumor were calculated for each time point [23].

2.6.

3.PET imaging

MicroPET scans were performed using a microPET Inveon rodent model scanner (Siemens Medical Solutions,USA).HT29tumor-bearing mice were injected with 3.7MBq of 68Ga e NOTA e OA e IONP in 200m L of saline via the tail vein and 20-min

static PET scans were performed.The images were reconstructed with a maximum a posteriori (MAP)algorithm with no attenuation or scatter correction.For each microPET scan,ROIs were drawn over the tumor by using the IRW (Inveon Research Workshop)on decay-corrected whole body axial and coronal images.The tumor uptake of 68Ga e NOTA e OA e IONP was calculated in terms of the percentage injected activity per gram (%ID/g)in the ROIs.

2.6.4.PET/MRI fusion imaging

For PET/MRI fusion imaging,we used the same mice bed without moving the mice.MR images were obtained ?rst and then PET images 60min post-injection.All the MRI data were processed in ParaVision 4.0(Bruker,Germany)software and were converted to the DICOM format for co-registration with PET images.For PET/MRI fusion imaging,we manually registered the PET and MR images with the IRW software.2.7.In vivo binding of iron oxide nanoparticles

The tumor tissues were separated from the tail veins of injected mice with 1mg/mL of IONP and/or OA-conjugated IONP for 4h,and then ?xed in 4%para-formaldehyde for 4h.The specimens were subsequently dehydrated in graded ethanol,embedded in paraf ?n,and cut into 5-m m sections on a Reichert microtome.The sections were deparaf ?nized and hydrated,and then rinsed in PBS.Subsequently they were stained with Perls ’Prussian blue staining solution for 30min,and then counterstained with 0.02%neutral red (Sigma).The staining results were observed under an inverted optical microscope (OLYMPUS IX81,Olympus Inc.,Japan).

3.Results

3.1.Preparation of OA derivatives

OA is known to have an inhibitory effect on colon cancer cells.Therefore,in this study,we conjugated OA with nanoparticles and a NOTA chelator for 68Ga radiolabeling (Fig.1)to improve the cancer targeting activity of our new imaging agent and to facilitate dual imaging with PET and MRI.FT-IR spectroscopy was used to con ?rm the binding of the OA moiety and the nanoparticles (Fig.2(a)).To compare the r 2relaxivities of the nanoparticles as MRI agents,the IONP and NOTA e OA e IONP were examined by 4.7T MRI.As shown in Fig.2(b),the relaxivities of the NOTA e OA e IONP and NOTA e IONP were 157s à1m M à1and 221s à1m M à1,respectively.The reason for the lower relaxivity of the NOTA e OA e IONP was the thickness of its coating.To clarify the particles sizes and coating thickness,we measured TEM and DLS.As shown in Fig.2(c)and (d),the TEM image revealed the shape and size distribution of the IONP,and the DLS graphs clearly showed that the NOTA e OA e IONP were bigger (66nm)than the IONP (16nm)in PBS buffer.In addition,when the NOTA e IONP was coupled with a hydrophobic molecule,such as OA,low relaxivity was induced.As a result,the NOTA e OA e IONP had a lower relaxivity than the IONP.

We also examined the long-term stability of the IONP and NOTA e OA e IONP in PBS buffer and 10%FBS/PBS buffer (Fig.3(a)and (b)).In PBS buffer,the IONP and NOTA e OA e IONP had an analogous average size of about 14nm and 60nm,respectively,over 7days at room temperature.The size in 10%FBS/PBS buffer was also measured after 24h at room temperature,but no signi ?cant changes (18nm and 67nm,respectively)were observed.These ?ndings show that the IONP and NOTA e OA e IONP are stable under biological conditions and may possibly avoid unwanted aggrega-tion and RES uptake in the

body.

Fig.1.Schematic of the hybrid PET/MR imaging agent created with an OA-conjugated nanoparticle.

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3.2.In vitro inhibition of cancer cells by iron oxide nanoparticles We found that the viability of HT-29cancer cells was dose-dependently decreased by OA-conjugated IONP in the XTT assay results (Fig.4).Furthermore,the inhibitory effect of OA-conjugated IONP was signi ?cantly increased after treatment with concentrations greater than 64m M .In contrast,the OA-negative control,that is,the IONP alone,had no effect on HT-29cancer cells over 24h.

3.3.Cellular uptake of iron oxide nanoparticles

We con ?rmed the HT29cellular uptake rates of IONP by Prus-sian blue staining assay.After treatment with IONP or OA e IONP,the cellular uptake of OA e IONP by HT-29cancer cells was signi ?cantly greater than that of IONP alone (Fig.5(a)).Speci ?cally,we found that the uptake of OA e IONP primarily occurred in the cellular membrane and in the intracellular surface area by a binding assay of OA e IONP with cells and/or cell lysates (Fig.5(b)).

To determine the tumor-targeting ability of the nanoparticle,we determined cancer cell uptake of radiolabeled nanoparticles (Fig.5(c)).The binding af ?nities of HT-29cells for 68Ga e NOTA e OA e IONP and 68Ga e NOTA e IONP were evaluated.HT-29cells were incubated with the radioligands for 1,2,and 4h.The cellular uptake of 68Ga e NOTA e OA e IONP at 1,2,and 4h was 3.08?0.06%,4.17?0.08%,and 5.59?0.18%,respectively.For IONP,the uptake rates were 2.45?0.08%,3.53?0.01%,and 5.16?0.05%,respectively.Both radiolabeled nanoparticles showed time-dependent

tumor

Fig.2.Characterization of the nanoparticles.a)Overlay IR spectra of OA and NOTA e OA e IONP.b)The r 2and 1/T 2values as a function of the Fe concentration of the NOTA e IONP and NOTA e OA e IONP nanoparticles.c)TEM image of IONP e OA e NOTA.d)Size distribution histograms of NOTA e IONP and NOTA e OA e IONP.

S.-m.Kim et al./Biomaterials 34(2013)8114e 81218117

uptake.However,the tumor uptake of 68Ga e NOTA e OA e IONP by HT-29cells was increased by 8e 26%compared with 68Ga e NOTA e IONP,that is,the nanoparticle without OA.3.4.In vivo PET/MR images

We also performed studies of male BALB/c nude mice bearing HT-29cells.We injected 68Ga e NOTA e OA e IONP or 68Ga e NOTA e IONP into a tail vein and then performed MRI scans.In the MR images taken with 68Ga e NOTA e IONP,the relative signal intensity of the liver was signi ?cantly decreased 5min after the injection (Fig.6(a)and (b)).However,the relative signal intensity of the tu-mor did not differ until 2h after the injection of 68Ga e NOTA e IONP without OA.When 68Ga e NOTA e OA e IONP was injected into the HT-29xenograft mice,the intensity pattern was similar to that of 68

Ga e NOTA e IONP in the liver and showed signi ?cant decreases at 5min post-injection (Fig.6(c)and (d).However,the tumor also showed a marked decreased within 5min and kept decreasing slowly until 25min;the highest peak occurred at 25min and this level was maintained for 80min after the injection of 68Ga e NOTA e OA e IONP.Therefore,68Ga e NOTA e OA e IONP appears to be suitable for use as a contrast agent for tumor diagnosis using MRI.After 60min,we performed a PET scan.In the PET images alone,the liver uptake was highest,which means the nanoparticle was cleared from the liver.The tumor also showed moderate uptake (%ID/g ?3.07?0.76),which indicates that the radiolabeled nanoparticle can be applied as a radioligand for PET (Fig.7).For the PET/MRI

fusion images of the tumor we manually registered the PET/MRI fusion images using the same FOV after injection of the dual function agent,68Ga e NOTA e OA e IONP.The two images were pre-cisely matched with respect to the tumor,which means that our new 68Ga e NOTA e OA e IONP nanoparticle can be used for tumor diagnosis using PET,MRI,or PET/MRI simultaneously.High quality tumor images and precise quanti ?cation of the tumor area were obtained from the PET/MRI fusion images.3.5.Histological study

After the imaging studies,we recon ?rmed the uptake of the nanoparticles by the HT29xenograft mice.The IONP staining re-sults showed that uptake of OA e IONP by tumor tissues was greater than the uptake of IONP (Fig.8).The OA-linked IONP induced apoptosis of tumor cells and in ?ltration of immune cells in the tumor,whereas IONP alone did not.Furthermore,the area of uptake of NOTA e OA e IONP was mostly around the vessels and periphery,and was concentrated in areas of the tumor populated by immune cells.Uptake of the negative control,IONP alone,was not or was only weakly detected in some regions of the vessels and in some peripheral regions on the tumor (Fig.8).4.Discussion

Both PET and MRI are powerful tools,but each still has some limitations with respect to tumor detection.Therefore,these

days

Fig.3.In vitro stability and proliferation of the nanoparticles.a)Long-term stability graphs showing the average size of the IONP and NOTA e OA e IONP nanoparticles in 10%FBS/PBS buffer (a)and PBS buffer (b)at room

temperature.

Fig.4.The cytotoxic effect of IONP and NOTA e OA e IONP on HT-29tumor cells.The viability of HT-29cancer cells was determined after treatment with IONP at several con-centrations (0.5,1,2,4,8,16,32,64,128,256,and 512m M )and/or with NOTA e OA e IONP.Survival of cancer cell lines was determined using the Cell Proliferation Reagent XTT (Sigma e Aldrich,USA).The experiment was repeated 3times in duplicate.

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fusion imaging modalities such as PET/MRI are required,and we have to develop imaging agents for use in these systems.Here,we present in vivo PET/MRI fusion images obtained with a hybrid im-aging agent,68Ga e NOTA e OA e IONP,which was targeted at the tumor and was derived from a natural compound,OA,which is known to have an inhibitory effect on cancer cells.

Oleanolic acid moiety binding to the nanoparticles was con ?rmed by FT-IR spectroscopy.However,it was dif ?cult to ?nd any evidence of the characteristic absorption bands of the IONP e OA e NOTA as the peaks overlapped.In spectrum a,the sharp peak at 1694cm à1was assigned to the carbonyl of OA and this carbonyl peak disappeared after the coupling with IONP.Therefore,we could indirectly ascertain that the IONP were coupled with the OA and NOTA by using 68Ga labeling and a cell uptake experiment [24].Moreover,we obtained different results from the XTT study and from a cell binding assay performed with and without OA.In OA-conjugated nanoparticles,the tumor targeting,uptake,and inhibition activity were higher than that observed in the nano-particles alone.

Regarding the size distribution of the nanoparticles,OA e NOTA e IONP was about 4-fold bigger than NOTA e IONP.The size of the nanoparticles may have affected their relaxivities;that is,the relaxivity of NOTA e OA e IONP was lower than that of NOTA e IONP.It is known that coating molecules can reduce their magnetic strength.In addition,hydrophobic coating molecules prevent ac-cess to water protons in magnetic nanoparticles [25e 27].

We found that the viability of the cancer cells was signi ?-cantly decreased in a dose-dependent manner when these were incubated with NOTA e OA e IONP,but no such effect was observed when the cells were incubated with IONP alone.These results clearly demonstrate that the viability of cancer cells was decreased by the OA.This natural compound is a member of the pentacyclic triterpenoid acids and many reports have noted that OA can induce apoptosis and inhibit the growth of cancer cells.In the cellular uptake experiment,we obtained evidence that OA increased cellular uptake of IONP in vitro and in vivo .Speci ?cally,in the cell lysate binding assay,we found that 4of the major bands of OA-linked IONP were bound with materials in the cell lysate.These materials may have included several receptors such as transforming growth factor (TGF-b 1)receptor,peroxisome proliferator-activated receptor g (PPAR g ),and insulin-related receptors,which are known to interact with pentacyclic tri-terpenoid acids,including OA.Moreover,TGF-b 1and PPAR g are strongly expressed in HT-29cancer cells.Thus,the high uptake of OA-linked IONP by the tumor tissue was probably due to their binding with TNF-b 1receptor and PPAR g .Further studies are necessary to determine whether such binding does in fact occur and whether it and related mechanisms can be applied in the design of therapeutic nanoparticles.

Hybrid PET/MR imaging was performed in living mice using a 4.7T animal MRI and microPET scanner with our new radiolabeled nanoparticle,which was developed to overcome the inherent limitations of each imaging modality.The in vivo data showed that PET and MRI fusion imaging was possible using our nanoparticle;the merged PET images of the tumor showed biochemical activity in the tumor tissue and the MRI images added clear morphological information relating to the tissue.Nanoparticles are of increasing interest in cancer diagnosis and therapeutic research because they have a variety of functions that can be used for biological,chem-ical,engineering,and medical applications,including

diagnosis

Fig.5.IONP and NOTA e OA e IONP cellular uptake and binding with cell lysate protein.a)Perls ’Prussian blue staining for iron in HT-29cells.HT-29cells (2?104cells/well)were seeded in 24-well plates and incubated for 3h with NOTA e IONP and NOTA e OA e IONP.b)SDS-PAGE of OA ligands from HT-29cell lysate proteins.IONP and NOTA e OA e IONP had captured some ligands of OA from puri ?ed whole protein extracted from HT-29.c)HT-29tumor cell uptake (%)of the radioligand at 1,2,and 4h with 68Ga e NOTA e IONP or 68Ga e NOTA e OA e IONP.

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and treatment of disease,due to their ability to link to biological molecules such as peptides,proteins,nucleic acids,and small-molecule ligands.Furthermore,to enable better detection of can-cer,nanoparticles can be developed for use as dual-modality cancer-targeting nanoparticle probes by linking these with radioligands for use in PET and MRI scans.Our ?ndings indicate that 68Ga e NOTA e OA e IONP is a powerful nanoparticle that can be used as a diagnostic agent for PET or MRI or PET/MRI fusion im-aging.Moreover,we intend to undertake further evaluations of its ability to induce tumor death and

apoptosis.

Fig.6.In vivo MR images.a)T2-weighted MR images of a mouse before (left,t ?0min)and after (right:t ?5min)the injection of the IONP solution into a tail vein.b)MR signals were derived from linear ?tting of plots (tumor/liver).c)T2-weighted MR images of a mouse before (left:t ?0min)and after (right:t ?5min)the injection of the OA e IONP solution into a tail vein.d)MR signals were derived from linear ?tting of plots (tumor/liver).The dramatic decrease in contrast signals in a mouse injected with OA e IONP was monitored by T2-weighted MRI after OA e IONP was injected into a tail

vein.

Fig.7.In vivo PET/MRI fusion images.PET and MR images of 68Ga e NOTA e OA e IONP administered to mice bearing HT-29xenografts (images were taken at 1h post-injection).The PET and MRI tumor images were registered for tumor diagnosis with 68Ga e NOTA e OA e IONP by IRW software.Arrows indicate the HT-29tumor.

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8120

5.Conclusion

In this study,we developed a new dual-modality cancer-targeting nanoparticle probe,68Ga e NOTA e OA e IONP,which is based on a natural compound and is very stable over time.68Ga e NOTA e OA e IONP enabled speci ?c detection of HT-29cancer cells in vitro as well as in vivo in xenograft tumor models using PET and MRI.The hybrid PET/MRI imaging agent can potentially be used not only for tumor diag-nosis and analysis of tumor functionality,but also for simultaneous tumor resolutionwith accurate quanti ?cation of the region of interest.Acknowledgments

This study was supported in part by a grant from the Korea Basic Science Institute (D33404)and by a National Research Foundation of Korea (NRF)grant funded by the Korean Government (MEST)(2012-0006388).References

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Fig.8.Histologic analysis of the HT-29xenograft cancer.The tumor tissues were stained using Perls ’Prussian blue staining solution after intravenous (i.v.)injection into a mouse with IONP and NOTA e OA e IONP.(?100,?200,original magni ?cation).

S.-m.Kim et al./Biomaterials 34(2013)8114e 81218121