MR

ARTICLE pubs.acs.org/bc

Oleyl-Chitosan Nanoparticles Based on a Dual Probe for Optical/MR Imaging in Vivo Chang-Moon Lee,†,‡,§,# DooRye Jang,†,‡,§ Jin Kim,|| Su-Jin Cheong,†,‡,§ Eun-Mi Kim,†,‡,§ Min-Hee Jeong,†,‡,§ Sun-Hee Kim,†,‡,§ Dong Wook Kim,†,‡,§ Seok Tae Lim,†,‡,§ Myung-Hee Sohn,†,‡,§ Yong Yeon Jeong,^ and Hwan-Jeong Jeong†,‡,§,* Department of Nuclear Medicine, ‡Research Institute of Clinical Medicine, §Cyclotron Research Center, #Institute for Medical Sciences, Chonbuk National University Medical School and Hospital, Jeonju, Jeonbuk 561-712, Republic of Korea Department of Advanced Chemicals, Chonnam National University, Gwangju 500-757, Republic of Korea ^ Department of Radiology, Chonnam National University Medical School, Gwangju 501-746, Republic of Korea

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ABSTRACT: Oleic acid-conjugated chitosan (oleyl-chitosan) is a powerful platform for encapsulating oleic acid-decorated iron oxide nanoparticles (ION), resulting in a good magnetic resonance imaging (MRI) probe. Oleyl-chitosan could selfassemble into core-shell structures in aqueous solution and provide the effective core compartment for loading ION. IONloaded oleyl-chitosan nanoparticles showed good enhanced MRI sensitivity in a MR scanner. Cy5.5 dye was accessed to the oleyl-chitosan conjugate for near-infrared (NIR) in vivo optical imaging. After intravenous injection of ION-loaded Cy5.5-conjugated oleyl-chitosan (ION-Cy5.5-oleyl-chitosan) nanoparticles in tumor-bearing mice, both NIRF and MR imaging showed the detectable signal intensity and enhancement in tumor tissues via enhanced permeability and retention (EPR) effect. Tumor accumulation of the nanoparticles was confirmed through ex vivo fluorescence images and Prussian blue staining images in tumor tissues. It is concluded that ION-Cy5.5-oleyl-chitosan nanoparticle is highly an effective imaging probe for detecting tumor in vivo.

’ INTRODUCTION Magnetic iron oxide nanoparticles (MNPs) are of great interest for in vivo biomedical applications, including magnetically triggered drug delivery, tissue engineering, and magnetic resonance molecular imaging.1,2 Successful application is highly dependent on the surface modification of the particles because bare MNPs have poor colloidal stability and are opsonized and sequestered by the reticuloendothelial system (RES), in particular, by Kupffer cells in the liver.3,4 Polymers, such as poly(ethylene glycol) (PEG), dextran, polyvinyl-pyrrolidone (PVP), poly(acrylic acid), and chitosan, are usually used as an MNP stabilizer to enhance dispersibility in an aqueous medium.5-9 Chitosan derived from the deacetylation of chitin consists of repeating units of glucosamine and N-acetyl-glucosamine. The biocompatibility and biodegradation of chitosan are ideal for biomedical applications. Moreover, chitosan can be tailored to specific chemical modifications, because it has both active amine and hydroxyl groups in its backbone. Chitosan introduced with a hydrophobic moiety can form nanosized self-aggregates in aqueous media.10-13 Long-chain fatty acids, such as oleic acid, linoleic acid, and palmitic acid, have been used as a hydrophobic moiety for the formation of chitosan micelles.14 Chitosanfatty acid conjugates form the nanomicelles of a hydrophobic core-hydrophilic shell structure in aqueous solution. The core r 2011 American Chemical Society

provides the environment to load hydrophobic drugs, and the shell allows colloidal stability of the particles. A stable colloidal dispersion of iron oxide nanoparticles (IONs) is prepared by coprecipitation or a thermal method using oleic acid as a surfactant.15,16 Therefore, an amphiphilic chitosan-fatty acid is useful for surface modification of oleic acid-decorated IONs. In a previous paper, we suggested that the amphiphilic chitosanlinoleic acid conjugate forms self-assembled nanoparticles spherical in shape that can effectively load surfactant-decorated IONs.10,17 We anticipated that a chitosan derivate conjugated with oleic acid has potential for use in IONs. Moreover, IONloaded oleyl-chitosan (ION-oleyl-chitosan) nanoparticles may be functionalized with molecules of interest, such as target ligands, and used as a good contrast agent for molecular imaging in vivo and in vitro. Of molecular imaging modalities, magnetic resonance imaging (MRI) offers excellent spatial resolution, unlimited depth penetration, and lack of harmful radiation. However, MRI has a low sensitivity to contrast agents. The limited sensitivity of MRI can be improved by combination with a near-infrared fluorescence (NIRF) optical imaging modality.18 NIRF imaging has poor Received: May 25, 2010 Revised: December 16, 2010 Published: January 18, 2011 186

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after evaporation at 50 C, 10 mL of oleyl-chitosan solutions, with concentrations ranging from 1.0 10-5 to 1.0 mg/mL, were added. Cy5.5-Conjugation on Oleyl-Chitosan. Oelyl-chitosan (50 mg) was dissolved in 50 mL 0.1 M sodium borate buffer at pH 8.5, and Cy5.5-NHS (0.1 mg) in DMSO (10 μL) was added. After stirring for 6 h at room temperature in the dark, the reaction mixture was dialyzed using a dialysis membrane (MWCO: 12 KDa, Spectrum Laboratories Inc.) for three days. The resulting Cy5.5-conjugated oleyl-chitosan (Cy5.5-oleyl-chitosan) was lyophilized in the dark and stored at 4 C before use. Preparation and Characterization of ION-Loaded Cy5.5Oleyl-Chitosan Nanoparticles. Cy5.5-conjugated oleyl-chitosan nanoparticles were prepared according to our previous method.10 Briefly, 10 mg Cy5.5-conjugated oleyl-chitosan was dissolved in 10 mL 0.1 M acetic acid solution under stirring. The solution was sonicated using a probe-type sonicator (VCX 750 Ultrasonic Processor, Sonics & Materials, Inc., Newtown, CT) at 100 W for 5 min. After having been synthesized by a seed growth method, 12 nm IONs were loaded into the Cy5.5-oleyl-chitosan nanoparticles, according to our previous report.24 IONs dispersed in 500 μL chloroform were added to the Cy5.5-oleylchitosan nanoparticle solution. The mixture was emulsified using a probe-type sonicator at 100 W for 10 min. The organic solvent was evaporated by stirring for 12 h at room temperature, and the pH of the solution was adjusted to 7.0 with 0.1 N NaOH solution. The size distribution of ION-loaded Cy5.5-oleyl-chitosan (IONCy5.5-oleyl-chitosan) nanoparticles was determined by dynamic light scattering (DLS) using a Microtrac UPA-150 particle size analyzer (Microtrac Inc., Montgomeryville, PA, USA; the instrument installed in Jeonju KBSI was used). The morphology of the ION-Cy5.5-oleyl-chitosan nanoparticles was evaluated using an H-7650 transmission electron microscope (TEM, Hitachi Ltd., Tokyo, Japan). Iron determination and loading efficiency for the Cy5.5-oleyl-chitosan nanoparticles were performed according to a previously reported method.25 Relaxivity of ION-Cy5.5-Oleyl-Chitosan Nanoparticles. T2 relaxivity was measured at 1.5 T via a clinical MRI scanner (GE Signa Exite Twin Speed, GE Health Care, Milwaukee, WI) at room temperature. T2-weighted scans of ION-Cy5.5-oleyl-chitosan naoparticles in water were performed with 2400 ms repetition time (TR) and echo times (TE) ranging from 20 to 200 ms. Relaxivity values were calculated using least-squares curve fitting of relaxation time versus iron concentration. In Vitro Cytotoxicity Study. U87MG cells and RAW cells were seeded in 96-well plates at a density of 1 104 cells per well in 100 μL medium containing 10% FBS. The plates were incubated overnight at 37 C. The culture medium was replaced with 100 μL medium containing 25-200 μg/mL of ION-Cy5.5-oleylchitosan nanoparticles. After 24 h, the cytotoxicity was evaluated by determining the viability of the cells using an XTT assay. After adding XTT solution to the wells, the cell viability of the cells was determined by measuring UV absorption at 450 nm on a plate reader. In Vivo NIRF Imaging Study. In vivo NIRF imaging was performed using an IVIS spectrum small-animal in vivo imaging system (Caliper Lifescience, Hopkinton, MA). After anesthetizing with 1.5-2% isoflurane, ION-Cy5.5-oleyl-chitosan nanoparticle solution (100 μL) was injected through the tail vein of U87MG-bearing mice. The imaging was performed at 1, 3, and 5 h postinjection, using the Cy5.5 filter with the following settings: exposure time (1 s), f/stop (2), binning (8), and field

spatial resolution and shows a lack of location of a threedimensional target molecule with anatomical resolution. On the other hand, NIRF imaging is more sensitive than MRI.19 Therefore, the combination of two NIRF optical and MR imaging modalities offers attractive synergistic advantages in molecular imaging for clinical diagnostic and therapeutic techniques.20 In this study, we describe the usefulness of oleyl-chitosan as a stabilizer of IONs. Furthermore, we investigate tumor accumulation of ION-oleyl-chitosan nanoparticles through the enhanced permeability and retention (EPR) effect in vivo for diagnostic applications using both NIRF optical and MR imaging.

’ EXPERIMENTAL PROCEDURES Regents and Chemicals. Chitosan (molecular weight: 50-80 KDa, deacetylation: 87%) was purchased from Kittolife Co. (Seoul, Korea). Fe(acac)3, oleic acid, oleylamine, 1,2-hexadecanediol, phenyl ether, N-hydroxysuccinimide (NHS, 97%), and 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). A monoreactive hydroxysuccinimide ester of Cy5.5 (Cy5.5-NHS) was purchased from GE Healthcare (Uppsala, Sweden). All other chemicals were of analytic grade and used directly without further purification. Tumor Xenograft Model. Female athymic nude mice (4 weeks old with weight 15 g) were obtained from Orient Bio, Inc. (Seoul, Korea). All animal experiments were performed according to the Institutional Animal Care and Use Committee for animal treatment of Chonbuk National University. Mice were anesthetized by subcutaneous injection of a mixture of ketamine (50 mg/kg body weight) and xylanzine (10 mg/kg body weight). A U87MG (glioblastoma) tumor xenograft model was prepared by subcutaneous injection into the right flank with 5 106 U87MG cells mixed in Matrigel (BD Bioscience, San Jose, CA, USA). Tumor-bearing mice were subjected to in vivo imaging studies when the tumor reached 0.7-1.0 cm in length. Synthesis and Characterization of Oleyl-Chitosan. Oelylchitosan was synthesized by a coupling reaction of the carboxyl group of oleic acid with the amine group of chitosan according to a previous method with slight modification.10,21 Chitosan (100 mg) was dissolved in 50 mL acetic acid solution (1%, v/v), and 8 mg oleic acid was dissolved in 10 mL ethanol. The two solutions were mixed at 80 C under stirring. After adding 27.4 mg EDC, the coupling reaction was carried out for 6 h at 80 C under stirring. The final solution was dialyzed against a 10% (v/v) ethanol solution using a dialysis membrane (MWCO: 12 KDa, Spectrum Laboratories Inc., Rancho Dominguez, CA) for three days. To remove ethanol from the product, dialysis was performed against distilled water for 24 h. Finally, the dialyzed product was lyophilized. The substitute degree (SD) of oleic acid to chitosan was measured by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum of the oleylchitosan conjugate was obtained using a 600 MHz spectrometer (JNM-ECA600, JEOL Ltd., Tokyo, Japan). The synthesized oleyl-chitosan was characterized by Fourier transform infrared (FT-IR) spectroscopy (Spectrum GX, Perkin-Elmer Inc., Waltham, MA). Measurement of Fluorescence Spectroscopy. The critical micelle concentration (CMC) of oleyl-chitosan was determined by fluorescence measurement using pyrene as a hydrophobic probe.22,23 Pyrene was dissolved in acetone (6.0 10-2 M), and 187

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Figure 1. Chemical structure of Cy5.5-oleyl-chitosan.

of view (12.8). At the end of the in vivo imaging, the mice were sacrificed, and the major organs were selected for ex vivo imaging. In Vivo MRI Study. U87MG-bearing mice were anesthetized with 1.5-2% isoflurane. ION-Cy5.5-oleyl-chitosan nanoparticle solution (100 μL) was intravenously injected into the tail vein of the mice. T2-weighted MR images were obtained with a 1.5 T clinical MR scanner (GE Signa Exite Twin-speed, GE Health Care, Milwaukee, WI), using an animal coil (4.3 cm Quadrature volume coil, Nova Medical System, Wilmington, DE). Fast spin-echo (FSE) T2-weighted MR imaging was performed with a repetition time/echo time of 4200/102 (ms/ms), flip angle of 90, echo train length of 10, field of view of 5 cm, section thickness of 2 mm, intersection gap of 0.2 mm, and a 256 160 matrix. The regions of interest (ROI) of tumors at identical locations were defined by one radiologist for quantitative analysis. The relative signal enhancement of the tumor, as compared to normal muscle, was calculated pre- and postinjection, using the following formula: [(Signal intensity of post - Signal intensity of pre)/Signal intensity of pre] 100. Prussian Blue Staining of Tumor Tissues. To confirm the accumulation in tumors of ION-Cy5.5-oleyl-chitosan nanoparticles, tumors were selected from animals at 3 h postinjection. The tumor tissues were fixed in 4% buffered formalin and then embedded in paraffin. After sectioning the tumor tissues with 4 μm thickness, the accumulation of ION-Cy5.5-oleyl-chitosan nanoparticles in the tumor was identified by Prussian blue staining. All micrographs were taken using a light microscope with a digital camera. Statistical Analysis. Quantitative data were expressed as mean ( SD. Means were compared by use of an independent samples t test. P values of less than 0.05 were considered statistically significant.

Figure 2. FT-IR spectra of (a) oleyl-chitosan conjugate and (b) chitosan.

were sharper in oleyl-chitosan. These results are evidence of the presence of oleic acid in the chitosan modification. Fluorescence Spectroscopy for CMC. Oleyl-chitosan nanoparticle formation in aqueous media was investigated using a fluorescence probe. The CMC value of oleyl-chitosan was examined using a dye solubilization method with pyrene (Figure 3). The intensity ratio of the first peak (I1) at 374 nm to the third peak (I3) at 384 nm of the pyrene emission spectra corresponding to oleyl-chitosan concentration was used to determine the CMC value. As shown in Figure 3B, the ratios of I1/I3 are nearly unchanged at low concentrations of oleyl-chitosan, whereas at higher concentrations, the ratios decreased, indicating self-aggregation of oleyl-chitosan. The CMC value of oleyl-chitosan was 0.112 mg/mL. Characterization of ION-Cy5.5-Oleyl-Chitosan Nanoparticles. Figure 4 shows the size distribution of ION-Cy5.5-oleylchitosan nanoparticles determined by DLS. The DLS results show that the diameter of the nanoparticles was 86.1 ( 45.6 nm. The TEM image shows clusters of IONs inside the core of an oleyl-chitosan nanoparticle (Figure 4b). ION-Cy5.5-oleyl-chitosan nanoparticles were fairy dispersed in distilled water and PBS buffer solution without any aggregation or precipitation. The loading efficiency of IONs loaded into the nanoparticles was 93.2%. Magnetic resonance sensitivity of ION-Cy5.5-oleyl-chitosan nanoparticles was measured using a clinical 1.5 T MRI scanner. Figure 5 shows a strong NIRF signal image and T2weighted images of the ION-Cy5.5-oleyl-chitosan nanoparticle solution at various concentrations. The T2 relaxivites of IONCy5.5-oleyl-chitosan nanoparticles linearly increased as the concentration of the nanoparticles increased. To examine the toxicity of ION-Cy5.5-oleyl-chitosan nanoparticles, the amount of viable cells was determined at the concentration range 25-200 μg/mL of the nanoparticles by the XTT assay. Figure 6 demonstrates that ION-Cy5.5-oleyl-chitosan nanoparticles exhibited little cytotoxicity to the two cells at relatively high concentration. The RAW cells and U87MG cells viabilities were 86.9 ( 3.5% and 88.5 ( 6.6% at 200 μg/mL of ION-Cy5.5-oleyl-chitosan nanoparticles, respectively. The encapsulation of ION with oleylchitosan nanoparticles effectively suppressed the cytotoxicity of iron particles. NIRF Imaging Study. NIRF imaging of U87MG-bearing mice was performed 1, 2, and 3 h after the intravenous injection of the ION-Cy5.5-oleyl-chitosan nanoparticles solution. As

’ RESULTS Characterization of Oleyl-Chitosan. The chemical structure of Cy5.5-conjugated oleyl-chitosan is shown in Figure 1. Oleylchitosan was synthesized by the coupling reaction of the carboxyl group of oleic acid with the amine group of chitosan in the presence of EDC. The degree of substitution (DS) of oleyl-chitosan was measured by 1H NMR spectroscopy. The number of oleic acid groups per 100 anhydroglucose units of chitosan was 4.14. The IR spectra of oleyl-chitosan and chitosan are shown in Figure 2. Specific peaks from the conjugation of oleic acid to chitosan were observed at 770 cm-1 for the CH alkyl group. Compared to the absorption of chitosan, the intensities of oleylchitosan at 1514 and 1452 cm-1 (which were assigned to CdC stretching) increased. The peaks at 1652 cm-1 for amide groups 188



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Figure 3. (a) Fluorescence spectra of pyrene in oleyl-chitosan nanoparticle solutions with various concentrations and (b) a plot of the fluorescence intensity ratio I1/I3 against the logarithm of oleyl-chitosan concentration.

Figure 4. (a) Size distribution and (b) TEM image of ION-Cy5.5-oleyl-chitosan nanoparticles. Inset in the graph shows the magnified TEM image of ION-Cy5.5-oleyl-chitosan nanoparticles.

Figure 5. (a) Bright and NIRF images of the ION-Cy5.5-oleyl-chitosan nanoparticle solution. (b) T2-weighted images of the ION-Cy5.5-oleylchitosan nanoparticle solution at various concentrations.

shown in Figure 7, some areas of fluorescence signal intensity were observed in the tumor, indicating accumulation of detectable amounts of ION-Cy5.5-oleyl-chitosan nanoparticles. The fluorescence signal intensity in the tumor tissue gradually increased up to 5 h postinjection and was clearly observed up to 1 day. Major organs of the mice were extracted 3.5 h after injection of the nanoparticles, and ex vivo NIRF imaging was performed (Figure 7E). The fluorescence signal intensity of the tumor tissue was significantly higher than that of normal muscle. The liver and kidney showed high fluorescence signal intensity. There was no accumulation of ION-Cy5.5-oleyl-chitosan nanoparticles in other organs, such as the heart, spleen, bone, or muscle.

Figure 6. In vitro cell viability of RAW cells and U87MG cells treated with the ION-Cy5.5-oleyl-chitosan nanoparticle solution by an XTT assay. (*p < 0.001, **p < 0.01).

MR Imaging Study and Prussian Blue Staining. T2weighted MR imaging of tumor-bearing mice using a clinical 1.5 T MRI scanner was performed at 1, 2, and 3 h after intravenous injection of ION-Cy5.5-oleyl-chitosan nanoparticles in PBS buffer solution. The T2-weighted images are presented in Figure 8. Before injection of the nanoparticles, the tumor appears 189



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Figure 7. In vivo NIRF images of U87MG-bearing mice. The images were obtained (a) before injection and at (b) 1 h, (c) 3 h, and (d) 5 h after injection of ION-Cy5.5-oleyl-chitosan nanoparticles. (e) Ex vivo NIRF images of major tissues excised from the mice at 5 h postinjection. (f) A quantification of the ex vivo tissues was recorded as average radiance (p/s/cm2/sr). All data are represented as mean ( SD (n = 3).

efficiency and sustained release of DOX, and efficiently delivered it to cancer cells. Although the potential of oleyl-chitosan nanoparticles as carriers for antitumor agents have been reported, the utility of oleyl-chitosan nanoparticles as a drug carrier and an imaging probe in vivo had not been reported. In this study, we prepared and characterized a dual imaging probe based on oleyl-chitosan nanoparticles for MRI and optical tumor imaging in vivo. Oleyl-chitosan can self-aggregate with the structure of the hydrophobic core and hydrophilic shell due to the hydrophobic oleic acid modification, as shown in Scheme 1. Oleyl-chitosan provides the hydrophobic core to load IONs and coupling site to conjugate dyes and targeting moieties. Cy5.5 was accessed for biodistribution and tumor accumulation of oleyl-chitosan through NIRF imaging, and IONs were encapsulated in the inner core of oleyl-chitosan nanoparticles for MRI in vivo (Scheme 1). The TEM image of ION-Cy5.5-oleylchitosan nanoparticles shows successful encapsulation of IONs into the core of the nanoparticles. Mean diameter of ION-Cy5.5-oleylchitosan nanoparticles determined by DLS is suitable for intravenous injection and passive targeting of tumors.26-28 It has been reported that the size of nanoparticles for tumor targeting based on the EPR effect is in the range of 200 nm or less.29 In generally, nanoparticles with mean diameter of 150-300 nm mainly accumulated in liver and spleen.30 To demonstrate the ability of ION-Cy5.5-oleyl-chitosan nanoparticles as a dual probe for optical/MR imaging, we performed the optical and MR phantom test, respectively. The strong NIRF signal enhancement of Cy5.5-labeled ION-oleyl-chitosan nanoparticles facilitates noninvasive optical imaging and ION clustering in the oleyl-chitosan nanoparticles dramatically increases T2 relaxivity than that of IONs.31 These phantom studies indicate that ION-Cy5.5-oleyl-chitosan nanoparticles can be potential as a dual probe for optical and MR imaging, simultaneously. The subsequent in vivo optical/MR imaging studies show the accumulation of ION-Cy5.5-oleyl-chitosan nanoparticles in the tumor, indicating the EPR effect-based tumor targeting ability.

Figure 8. In vivo T2-weighted MR images of U87MG-bearing mice (a) before and at (b) 1 h, (c) 2 h, and (d) 3.5 h after injection of ION-Cy5.5oleyl-chitosan nanoparticles on a 1.5 T MR scanner. Yellow arrows in the dark area of the tumor tissue indicate accumulation of ION-Cy5.5-oleylchitosan nanoparticles. (e) Prussian blue staining image of tumor tissues dissected from a mouse at 5 h postinjection.

as a hyperintense lesion. The relative signal intensity of the ROI in the tumor area was significantly decreased after injection of the nanoparticles. The signal intensity enhancement in the tumor areas was approximately 16.6% at 1 h, 22.4% at 2 h, and 15.9% at 3.5 h. At 3 h postinjection, a noticeable darkening was seen in the peripheral area of the tumor tissue (see the arrows in Figure 8D). The accumulation of ION-Cy5.5-oleyl-chitosan nanoparticles in the tumor tissues was assessed histologically using Prussian blue staining. As shown in Figure 8E, iron particles were stained with blue color in the tumor tissue slices injected with ION-Cy5.5oleyl-chitosan nanoparticles.

’ DISCUSSION Oleyl-chitosan nanoparticles have been investigated extensively for drug and gene delivery applications.12,22 For example, Zhang et al. 12 developed self-assembled nanoparticles based on oleyl-chitosan, which were loaded with doxorubicin (DOX). In their study, oleyl-chitosan nanoparticles showed high encapsulation 190



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Scheme 1a

a

(a) Diagram of Cy5.5 and oleic acid-conjugated chitosan, (b) self-assembled nanoparticles for the conjugate, and (c) ION-Cy5.5-oleyl-chitosan nanoparticles.

The accumulation in the liver is related to the reticuloendotheial system (RES).32 In general, most nanoparticles are quickly sequestered by phagocyte cells from the blood after injection into the body. The high fluorescence signals in the kidneys indicate that ION-Cy5.5-oleyl-chitosan nanoparticles are cleared via the renal route.33 The clearance could be confirmed through the NIRF signal from the urine and Prussian blue staining of the kidney tissues. Furthermore, ex vivo NIRF imaging results clearly show that ION-Cy5.5-oleyl-chitosan nanoparticles can successfully accumulate in tumor tissue. These results indicate that ION-Cy5.5-oleyl-chitosan nanoparticles were successfully accumulated in the tumor tissues of tumor-bearing mice by the EPR effect, resulting in efficient diagnosis of the tumors. In conclusion, we have introduced oleyl-chitosan nanoprobe encapsulating IONs for tumor imaging in vivo. The oleylchitosan conjugates were able to form self-assembled nanoparticles, as well as provide an effective loading compartment for IONs. The feasibility of ION-Cy5.5-oleyl-chitosan nanoparticles as an NIRF probe and MR contrast agent was examined in tumor-bearing mice. ION-Cy5.5-oleyl-chitosan nanoparticles could detect tumors in vivo via the EPR effect using both NIRF and MR imaging. This nanoparticle may be used as a probe for tumor diagnosis, and we plan to develop a novel nanodrug for simultaneous diagnosis and therapy of tumors.

(2) Lu, A. H., Salabas, E. L., and Sch€uth, F. (2007) Magnetic nanoparticles: synthesis, protection, functionalization, and application. Angew. Chem., Int. Ed. 46, 1222–1244. (3) Hu, F., Neoh, K. G., Cen, L., and Kang, E. T. (2006) Cellular response to magnetic nanoparticles “PEGylated” via surface-initiated atom transfer radical polymerization. Biomacromolecules 7, 809–816. (4) Kohler, N., Fryxell, G. E., and Zhang, M. (2004) A bifunctional poly(ethylene glycol) silane immobilized on metallic oxide-based nanoparticles for conjugation with cell targeting agents. J. Am. Chem. Soc. 126, 7206–7211. (5) Larsen, E. K., Nielsen, T., Wittenborn, T., Birkedal, H., VorupJensen, T., Jakobsen, M. H., Ostergaard, L., Horsman, M. R., Besenbacher, F., Howard, K. A., and Kjems, J. (2009) Size-dependent accumulation of PEGylated silane-coated magnetic iron oxide nanoparticles in murine tumors. ACS Nano 3, 1947–1951. (6) Lee, C. M., Jeong, H. W., Kim, E. M., Cheong, S. J., Park, E. H., Kim, D. W., Lim, S. T., and Sohn, M. H. (2009) Synthesis and characterization of iron oxide nanoparticles decorated with carboxymethyl Curdlan. Macromol. Res. 17, 133–136. (7) Lee, H. Y., Lim, N. H., Seo, J. A., Yuk, S. H., Kwak, B. K., Khang, G., Lee, H. B., and Cho, S. H. (2006) Preparation and magnetic resonance imaging effect of polyvinylpyrrolidone-coated iron oxide nanoparticles. J. Biomed. Mater. Res., Part B 79, 142–150. (8) Liang, Y. Y., and Zhang, L. M. (2007) Bioconjugation of papain on superparamagnetic nanoparticles decorated with carboxymethylated chitosan. Biomacromolecules 8, 1480–1486. (9) Lin, C. L., Lee, C. F., and Chiu, W. Y. (2005) Preparation and properties of poly(acrylic acid) oligomer stabilized superparamagnetic ferrofluid. J. Colloid Interface Sci. 291, 411–420. (10) Lee, C. M., Jeong, H. J., Kim, S. L., Kim, E. M., Kim, D. W., Lim, S. T., Jang, K. Y., Jeong, Y. Y., Nah, J. W., and Sohn, M. H. (2009) SPION-loaded chitosan-linoleic acid nanoparticles to target hepatocytes. Int. J. Pharm. 371, 163–169. (11) Xing, K., Chen, X. G., Liu, C. S., Cha, D. S., and Park, H. J. (2009) Oleoyl-chitosan nanoparticles inhibits Escherichia coli and Staphylococcus aureus by damaging the cell membrane and putative binding to extracellular or intracellular targets. Int. J. Food Microbiol. 132, 127–133. (12) Zhang, J., Chen, X. G., Peng, W. B., and Liu, C. S. (2008) Uptake of oleyl-chitosan nanoparticles by A549 cells. Nanomedicine 4, 208–214. (13) Park, K., Hong, H. Y., Moon, H. J., Lee, B. H., Kim, I. S., Kwon, I. C., and Rhee, K. (2008) A new atherosclerotic lesion probe based on hydrophobically modified chitosan nanoparticles functionalized by the atherosclerotic plaque targeted peptides. J. Controlled Release 128, 217–223. (14) Wydro, P., Krajewska, B., and Hac-Wydro, K. (2007) Chitosan as a lipid binder: a langmuir monolayer study of chitosan-lipid interactions. Biomacromolecules 8, 2611–2617. (15) Song, H. T., Choi, J. S., Huh, Y. M., Kim, S., Jun, Y. W., Suh, J. S., and Cheon, J. (2005) Surface modulation of magnetic nanocrystals in the development of highly efficient magnetic resonance probes for intracellular labeling. J. Am. Chem. Soc. 127, 9992–9993.

’ AUTHOR INFORMATION Corresponding Author

*Hwan-Jeong Jeong, MD, PhD, Department of Nuclear Medicine, Chonbuk National University Hospital, 634-18, Geumam-2 dong, Dukjin-gu, Jeonju, Jeonbuk, Republic of Korea 561712. Phone: 82-63-250-1674. Fax: 82-63-250-1676. E-mail: [email protected].

’ ACKNOWLEDGMENT This study was supported by a grant from the National R&D Program for Cancer Control, Ministry of Health, Welfare and Family Affairs, Republic of Korea (0620220 and 0720420). This work was also supported by the National Research Foundation of Korea Grant funded by the Korean Government (Ministry of Education, Science and Technology). [NRF-2010-359-D00004]. ’ REFERENCES (1) Gupta, A. K., and Gupta, M. (2005) Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 26, 3995–4021. 191



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(16) Sun, J., Zhou, S., Hou, P., Yang, Y., Weng, J., Li, X., and Li, M. (2007) Synthesis and characterization of biocompatible Fe3O4 nanoparticles. J. Biomed. Mater. Res., Part A 80, 333–341. (17) Cheong, S. J., Lee, C. M., Kim, S. L., Jeong, H. J., Kim, E. M., Park, E. H., Kim, D. W., Lim, S. T., and Sohn, M. H. (2009) Superparamagnetic iron oxide nanoparticles-loaded chitosan-linoleic acid nanoparticles as an effective hepatocyte-targeted gene delivery system. Int. J. Pharm. 372, 169–176. (18) Jennings, L. E., and Long, N. J. (2009) `Two is better than one’probes for dual-modality molecular imaging. Chem. Commun. 28, 3511– 3524. (19) Ke, J. H., Lin, J. J., Carey, J. R., Chen, J. S., Chen, C. Y., and Wang, L. F. (2010) A specific tumor-targeting magnetofluorescent nanoprobe for dual-modality molecular imaging. Biomaterials 31, 1707– 1715. (20) Nam, T., Park, S., Lee, S. Y., Park, K., Choi, K., Song, I. C., Han, M. H., Leary, J. J., Yuk, S. A., Kwon, I. C., Kim, K., and Jeong, S. Y. (2010) Tumor targeting chitosan nanoparticles for dual-modality optical/MR cancer imaging. Bioconjugate Chem. 21, 578–582. (21) Du, Y. Z., Wang, L., Yuan, H., Wei, X. H., and Hu, F. Q. (2009) Preparation and characterization of linoleic acid-grafted chitosan oligosaccharide micelles as a carrier for doxorubicin. Colloids Surf., B 69, 257–263. (22) Hu, F. Q., Wu, X. L., Du, Y. Z., You, J., and Yuan, H. (2008) Cellular uptake and cytotoxicity of shell crosslinked stearic acid-grafted chitosan oligosaccharide micelles encapsulating doxorubicin. Eur. J. Pharm. Biopharm. 69, 117–125. (23) Li, Y. Y., Chen, X. G., Liu, C. S., Cha, D. S., Park, H. J., and Lee, C. M. (2007) Effect of the molecular mass and degree of substitution of oleoylchitosan on the structure, rheological properties, and formation of nanoparticles. J. Agric. Food Chem. 55, 4842–4847. (24) Lee, C. M., Jeong, H. J., Kim, E. M., Kim, D. W., Lim, S. T., Kim, H. T., Park, I. K., Jeong, Y. Y., Kim, J. W., and Sohn, M. H. (2009) Superparamagnetic iron oxide nanoparticles as a dual imaging probe for targeting hepatocytes in vivo. Magn. Reson. Med. 62, 1440–1446. (25) Lee, C. M., Jeong, H. J., Cheong, S. J., Kim, E. M., Kim, D. W., Lim, S. T., and Sohn, M. H. (2010) Prostate cancer-targeted imaging using magnetofluorescent polymeric nanoparticles functionalized with bombesin. Pharm. Res. 27, 712–721. (26) Li, S. D., and Huang, L. (2008) Pharmacokinetics and biodistribution of nanoparticles. Mol. Pharmaceutics 5, 496–504. (27) Moghimi, S. M., Hunter, A. C., and Murray, J. C. (2001) Longcirculating and target-specific nanoparticles: Theory to practice. Pharmacol. Rev. 53, 283–318. (28) Zhao, Z., He, M., Yin, L., Bao, J., Shi, L., Wang, B., Tang, C., Yin, C. (2009) Biodegradable nanoparticles based on linoleic acid and poly(beta-malic acid) double grafted chitosan derivatives as carriers of anticancer drugs. Biomacromolecules 10, 565-572. (29) Kim, K., Kim, J. H., Park, H., Kim, Y. S., Park, K., Nam, H., Lee, S., Park, J. H., Park, R. W., Kim, I. S., Choi, K., Kim, S. Y., Park, K., Kwon, I. C. (2010) Tumor-homing multifunctional nanoparticles for cancer theragnosis: Simultaneous diagnosis, drug delivery, and therapeutic monitoring. J. Controlled Release 146, 219-227. (30) Moghimi, S. M., Hunter, A. C., and Murray, J. C. (2001) Longcirculating and target-specific nanoparticles: theory to practice. Pharmacol. Rev. 53, 283–318. (31) Ai, H., Flask, C., Weinberg, B., Shuai, X., Pagel, M. D., Farrell, D., Duerk, J., and Gao, J. (2005) Magnetite-loaded polymeric micelles as ultrasensitive magnetic-resonance probes. Adv. Mater. 17, 1949–1952. (32) Ferrucci, J. T., and Stark, D. D. (1990) Iron oxide-enhanced MR imaging of the liver and spleen: review of the first 5 years. Am. J. Roentgenol. 155, 943–950. (33) Chen, X., Xie, J., Xu, H., Behera, D., Michalski, M. H., Biswal, S., Wang, A., and Chen, X. (2009) Triblock copolymer coated iron oxide nanoparticle conjugate for tumor integrin targeting. Biomaterials 30, 6912–6919.

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