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Tumor Targeting Chitosan Nanoparticles for Dual-Modality Optical/MR Cancer Imaging Taehwan Nam,†,‡,§ Sangjin Park,†,§ Seung-Young Lee,§ Kyeongsoon Park,§ Kuiwon Choi,§ In Chan Song,| Moon Hee Han,| James J. Leary,⊥ Simseok Andrew Yuk,§,# Ick Chan Kwon,§ Kwangmeyung Kim,*,§ and Seo Young Jeong*,‡ Department of Life and Nanopharmaceutical Science, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul, 130-701, Korea, Biomedical Research Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul, 136-791, Korea, Department of Radiology, Seoul National University College of Medicine, 101 Daehangno, Chongno-Ko, 110-744, Korea, and Weldon School of Biomedical Engineering and School of Chemical Engineering, Purdue University, West Lafayette, Indiana. Received September 16, 2009; Revised Manuscript Received January 26, 2010

We report tumor targeting nanoparticles for optical/MR dual imaging based on self-assembled glycol chitosan to be a potential multimodal imaging probe. To develop an optical/MR dual imaging probe, biocompatible and water-soluble glycol chitosan (Mw ) 50 kDa) were chemically modified with 5β-cholanic acid (CA), resulting in amphiphilic glycol chitosan-5β-cholanic acid conjugates (GC-CA). For optical imaging near-infrared fluorescence (NIRF) dye, Cy5.5, was conjugated to GC-CA resulting in Cy5-labeled GC-CA conjugates (Cy5.5-GC-CA). Moreover, in order to chelate gadolinium (Gd(III)) in the Cy5.5-GC-CA conjugates, 1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid (DOTA) was directly conjugated in Cy5.5-GC-CA. Finally, the excess GdCl3 was added to DOTA modified Cy5.5-GC-CA conjugates in distilled water (pH 5.5). The freshly prepared Gd(III) encapsulated Cy5.5-GC-CA conjugates were spontaneously self-assembled into stable Cy5.5 labeled and Gd(III) encapsulated chitosan nanoparticles (Cy5.5-CNP-Gd(III)). The Cy5.5-CNP-Gd(III) was spherical in shape and approximately 350 nm in size. From the cellular experiment, it was demonstrated that Cy5.5-CNP-Gd(III) were efficiently taken up and distributed in cytoplasm (NIRF filter; red). When the Cy5.5-GC-Gd(III) were systemically administrated into the tail vein of tumor-bearing mice, large amounts of nanoparticles were successfully localized within the tumor, which was confirmed by noninvasive near-infrared fluorescence and MR imaging system simultaneously. These results revealed that the dual-modal imaging probe of Cy5.5-CNP-Gd(III) has the potential to be used as an optical/MR dual imaging agent for cancer treatment.

Recently, nanotechnology and molecular imaging have been combined to generate multifunctional nanoparticles that simultaneously facilitate early-stage cancer diagnosis, target drug delivery with minimal toxicity, and monitor cancer treatment (1-3). In particular, various multimodal nanoparticle imaging probes have been tested to overcome the limitations of either imaging method when used alone (4, 5). The combination of magnetic resonance (MR) imaging and near-infrared fluorescence (NIRF) optical imaging for cancer diagnosis have several attractive attributes that have driven the development of molecular imaging strategies for these modalities (6-8). The major advantage of NIRF optical imaging is the capability of visualizing target biological molecules (peptides, drugs, DNA, siRNA, proteins, etc.) in vivo with a higher sensitivity (9-15). However, the spatial resolution of NIRF optical imaging is not enough to show the location of a threedimensional target molecule with anatomical resolution. MR imaging is one of the imaging techniques used to obtain threedimensional information. Besides the excellent spatial resolution, one limitation of MRI is its low sensitivity to contrast agents, * Corresponding author. Tel: +82-2-958-5912; Fax: +82-2-9585909; E-mail: [email protected] (K. Kim). Tel: +82-2-966-3885; Fax: +82-2-961-0356; E-mail: [email protected] (S. Y. Jeong). † These authors contributed equally to this paper. ‡ Kyung Hee University. § Korea Institute of Science and Technology. | Seoul National University College of Medicine. ⊥ Weldon School of Biomedical Engineering, Purdue University. # School of Chemical Engineering, Purdue University.

which are important for the detection of cellular or molecular changes on a nanomolar scale. Therefore, the limited sensitivity of MR imaging can be overcome by combination with NIRF optical imaging methods. Of note, polymeric nanoparticles are known to accumulate at the solid tumor site by the so-called enhanced permeation and retention (EPR) effect, resulting in efficient accumulation in solid tumor tissues (16). Therefore, polymeric nanoparticles have been applied as effective multifunctional nanoparticles capable of enhancing the diagnostic and therapeutic efficacy of imaging probes and therapeutic agents in cancer treatment. This is due to the fact that polymeric nanoparticles exhibited ideal in vivo characteristics, such as biocompatibility and biodegradability, and prolonged circulation time the bloodstream. For example, Doiron et al. recently reported optical/T1 dual imaging agent based on PLGA which encapsulated Gd(III) and rhodamine (17). However, it did not describe biodistribution or in vivo tumor targeting. As an optical/T2 dual imaging agent, Yu et al prepared polymer-coated iron oxide nanoparticles with drug loading (18). Doxorubicin was used as an anticancer agent that exhibited fluorescence as an optical imaging agent. It efficiently targeted and showed antitumor effects in vivo. However, the imaging probe was still distributed in other major organs such as the liver. Herein, we introduce tumor targeting chitosan nanoparticles for optical/MR dual imaging that was based on polymeric nanoparticle technology and molecular imaging. Previously, we have developed hydrophobically modified glycol chitosan nanoparticles (HGC) as novel nanosized drug carriers in cancer

10.1021/bc900408z  2010 American Chemical Society Published on Web 03/04/2010

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Scheme 1. (a) Chemical Structure of Cy5.5-CNP-Gd(III) and (b) Schematic Illustration of Self-Assembled Chitosan Nanoparticles (Blue Color, Glycol Chitosan Shell; White Color, Inner Part of Chitosan Nanoparticle) and Tumor Targeting for Dual-Modal NIRF Optical/ MR Imaging Probes

treatment that can self-assemble in aqueous solution to form stable nanoparticles (19-21). Selective accumulation of HGC nanoparticles in the tumor interstitium by the EPR effect was exhibited when systemically administered to deliver various types of therapeutic agents (19, 21). To make new optical/MR dual imaging agents, we designed tumor targeting chitosan-based nanoparticles (CNPs) containing dual imaging agents (NIRF dye, Cy5.5 (20), and gadolinium (Gd(III)) ions) (16, 22, 23) (Scheme 1). The tumor targeting profile and cancer imaging potential of dual imaging probes of chitosan nanoparticles were evaluated in tumor-bearing mice. To make optical/MR dual imaging nanoparticles biocompatible and water-soluble, glycol chitosan (GC, Mw ) 50 kDa) was chemically modified with 5β-cholanic acid (CA) in the presence of EDC and NHS, which promoted amide linkage formation between the amine group of chitosan and carboxyl acid of CA, resulting in GC-CA (24-26). A colloidal titration method showed that GC-CA had an average molecular weight of 54 kDa, indicating that 11 ( 2 of CA molecules were chemically conjugated to GC (20). For the optical/MR dualmodal cancer imaging, 0.5 ( 0.1 of NIRF dye, monohydroxysuccinimide Cy5.5-NHS, was conjugated to GC-CA (Cy5.5GC-CA). In order to chelate Gd(III) in the conjugates, monoreactive hydroxysuccinimide-DOTA was directly conjugated to the amine groups in the Cy5.5-GC-CA. The degrees of conjugation were in the range from 13 to 45, confirmed by 1 H NMR (Table 1). Finally, the excess of GdCl3 was added to DOTA modified Cy5.5-GC-CA in distilled water (pH 5.5). After the reaction, EDTA tetrasodium salt was used to remove the unreacted Gd(III) ions. The weight ratio of Gd(III) in the conjugates was maximized to approximately 6 ( 0.28%, measured by energy dispersive spectroscopy (EDS) and inductively coupled plasma-atomic emission spectrometer (ICPAES) (Table 1; see Supporting Information). In this study, maximum Gd(III) encapsulated Cy5.5-labled chitosan nanopar-

Table 1. Characterization of the Weight Ratio of Gd(III) in CNPs sample

Ds of DOTAa

wt (%) of Gd(III)

diameter (nm)

Cy5.5-CNPs Cy5.5-CNPs-[Gd(III)-2%] Cy5.5-CNPs-[Gd(III)-4%] Cy5.5-CNPs-[Gd(III)-6%]

13 34 45

2.39 4.22 6.28

250 ( 18 260 ( 28 320 ( 35 350 ( 42

a

Degree of substitution of the number of DOTA per on CNPs.

ticles (Cy5.5-CNP-Gd(III); 6.28 wt %) were used to analyze their characteristics in vitro and in vivo. The dried Gd(III) encapsulated chitosan conjugates, when dissolved in aqueous solutions, spontaneously self-assembled into stable Cy5.5 labeled and Gd(III) encapsulated chitosan nanoparticles (Cy5.5-CNP-Gd(III)). Dynamic light scattering and transmission electron microscopy (TEM) analyses revealed that Cy5.5-CNP-Gd(III) were spherical and approximately 350 nm in size (Figure 1a). In phosphate buffered saline (PBS) at 37 °C, the Cy5.5-CNP-Gd(III) remained dispersed and maintained their original nanoparticle size for up to 40 days, indicating the excellent stability of nanoparticle probes in aqueous conditions (Figure 1b). Also, Cy5.5-CNP-Gd(III) were rapidly taken up within one hour by HeLa cells expressing a green fluorescence protein-tagged histone 2B (HeLa H2B-GFP), but free Cy5.5 molecules were not taken up by the cells within same time. Of note, most of the internalized particles were found in the cytoplasm (NIRF filter; red), not in the GFP-labeled nuclear compartment (FITC filter; green) (Figure 1c). This rapid cellular uptake characteristic of Cy5.5-CNP-Gd(III) facilitated delivery of anticancer drugs and imaging agents into the cytoplasmic compartment of target tumor cells. Moreover, Cy5.5-CNP-Gd(III) exhibited almost 100% of cell viability (Figure 1d), even at the relative high concentration up to 250 µg/mL. To demonstrate the ability of Cy5.5-CNP-Gd(III) as an optical/MR dual-modal imaging probe, we obtained the NIRF

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Figure 1. (a) Average size of Cy5.5-CNP-Gd(III) in PBS (pH 7.4, 37C), confirmed with dynamic light scattering. (b) In vitro stability of Cy5.5CNP-Gd(III) in PBS for 40 days. (c) Cellular uptake profiles of free Cy5.5 and Cy5.5-CNP-Gd(III) in a cell culture system. Each sample was incubated with HeLa H2B-GFP cells for 1 h (original magnification × 400). (d) Cell viability of Cy5.5-CNP-Gd(III) in SCC7 cells for 1 day.

Figure 2. (a) Bright and NIRF images of the Cy5.5-CNP-Gd(III) in PBS. (b) T1-weighted spin-echo images of Cy5.5-CNP-Gd(III) in acidic (pH 5.2) and physiological condition (pH7.4). (c) T1-weighted spin-echo MR image (1.5T) showing bright MR signals of DOTEM and Cy5.5-CNP-Gd(III) in PBS.

optical and MR images from the optical or MR phantom test, respectively. Cy5.5-CNP-Gd(III) solution (1 mg/mL in PBS) produced a strong NIRF signal when we used a Cy5.5 filter set (Figure 2a), facilitating noninvasive imaging of the nanoparticles in live animals. The bright signal enhancement in the T1weighted MR images revealed a progressive increase as the concentration of Cy5.5-CNP-Gd(III) increased. In particular, they showed the same T-1 weight MR image at the different pH conditions, indicating the stability of chelated Gd(III) ions in the nanoparticles (Figure 2b). As a comparison, we obtained MR images of commercialized MR contrast agent DOTAREM. The contrast efficiency of Cy5.5-CNP-Gd(III) was less than that of DOTAREM, at the higher Gd(III) concentration (>2.0 × 10-3 mM) (Figure 2c). It is deduced that the encapsulated Gd(III) in the hydrophobic inner part of nanoparticles decreased the

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Figure 3. In vivo time-dependent biodistribution of Cy5.5-CNP-Gd(III) in SCC7 tumor-bearing mice (diameter ) 8-10 mm). (a) In vivo timedependent whole body imaging after i.v. injection of Cy5.5-CNP-Gd(III) (5 mg/kg). (b) Ex vivo images of normal organs (liver, lung, spleen, heart, and kidney) and tumors excised at 1 day postinjection. (c) A quantification of in vivo biodistribution of Cy5.5-CNP-Gd(III) was recorded as total photon counts per centimeter squared per steradian (p/s/cm2/sr) per each excised organ at 1 day postinjection. All data represent mean ( s.e. (d) NIRF microscopic images of tumors excised from Cy5.5 (5 mg/kg)-labeled CNPs-treated SCC7 tumor-bearing mice. The NIRF microscopic image of each sample was viewed using an NIRF microscope with Cy5.5 filter set. Original magnification is × 100.

relaxivity of Gd(III), resulted in the reduction of accessibility in aqueous condition. Interestingly, it retained higher contrast signal at the lower concentration. This phantom study demonstrated the potential value of Cy5.5-CNP-Gd(III) as a contrast agent for optical and MR imaging, simultaneously. We subsequently monitored the time-dependent in vivo biodistribution of Cy5.5-CNP-Gd(III) after intravenous injection into nude mice bearing pectoral subcutaneous murine squamous carcinoma cells (SCC7) tumors of about 8-10 mm in diameter by using Kodak Image Station; with a special C-mount lens and Cy5.5 bandpass emission filter (Figure 3a). Cy5.5-CNP-Gd(III)-treated mice (5 mg/kg) presented a strong NIRF signal throughout the whole body within 6 h of injection, indicating that the nanoparticles rapidly circulated in the bloodstream. From the NIRF optical images, we could delineate subcutaneous tumors from the surrounding background tissue at 6 h postinjection, and they exhibited a maximum NIRF signal beginning at 12 h postinjection. It should be noted that the tumors maintained this maximal NIRF intensity for 3 days, but the NIRF signal persisted for up to 5 days postinjection, with a gradual decrease in signal in the tumor. Of note, ex vivo NIRF images of excised tissues (liver, lung, kidney, heart, and the tumors) provided substantial evidence for the EPR effect-based tumor targeting ability of Cy5.5-CNP-Gd(III) in SCC7 tumor-bearing mice (Figure 3b) at 1 day postinjection of Cy5.5-CNP-Gd(III). The image revealed that Cy5.5-CNP-Gd(III) were mainly taken up by the tumor tissues, whereas their uptake in normal tissues was not significant. Furthermore, the NIRF total photon counts per gram of each organ from the tumor tissues were 3- to 7-fold higher than those from other organs, providing decisive evidence that Cy5.5-CNP-Gd(III) specifically targeted tumor tissues (Figure 3c). Via microscale images, Cy5.5-CNP-Gd(III) initially localized in the peripheral region of tumors after 6 h postinjection in mice, and the

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was not enough to clearly delineate the exact tumor site (Figure 4b). As NIRF optical imaging offers high sensitivity and the prospect of real-time intraoperative monitoring of tumor biopsy, on the other hand, MR imaging offers excellent spatial resolution and intrinsic soft-tissue contract. Thus, the combination of NIRF optical imaging and MR imaging for cancer imaging may overcome the limitations of either imaging method when used alone. In conclusion, we prepared tumor targeting nanoparticles for optical/MR dual imaging that was based on self-assembled glycol chitosan nanoparticles. For the optical/MR dual imaging, Cy5.5 as NIRF and DOTA to chelate Gd(III) were directly conjugated in the chitosan conjugates. In tumor-bearing mice, Cy5.5-labeled and Gd(III) ion-chelated chitosan nanoparticles showed long and stable blood circulation characteristics, and they demonstrated excellent tumor targeting ability based on the EPR effects. More importantly, the combination of NIRF optical imaging and MR imaging for cancer imaging not only offered high sensitivity, but also presented excellent spatial resolution of target tumor tissue. Therefore, Cy5.5-CNP-Gd(III) has the potential to be used as optical/MR dual imaging agents for cancer imaging.

ACKNOWLEDGMENT Figure 4. Tumor target specificity of Cy5.5-CNP-Gd(III) in SCC7 tumor-bearing mice (diameter ) 8-10 mm). (a) Coronal slices of T1weighted MR images of Cy5.5-CNP-Gd(III) in SCC7 tumor-bearing mice at 0 and 1 h postinjection. Red circles indicate that SCC-7 tumor tissue. (b) Both NIRF and T1-weighted MR images were simultaneously observed after 1 day postinjection of Cy5.5-CNP-Gd(III) (5 mg/kg).

maximum NIRF intensity was observed throughout the entire tumor after 12 h postinjection (Figure 3d). All images were reproducible and revealed the feasibility as a targeted optimal imaging probe for in vivo cases. On the basis of this imaging data, our glycol chitosan nanoparticles showed higher tumor homing ability in tumor-bearing mice. Until the exact mechanism of the higher tumor homing ability is completely evaluated, the unique characteristics of the glycol chitosan nanoparticles that we observed in vitro (i.e., stability in blood, deformability, and rapid cellular uptake) may have contributed significantly to their tumor targeting by the EPR effect in vivo (19, 24-26). Furthermore, when the Cy5.5-CNPGd(III) was injected into the tumor-bearing mice, it presented a prolonged blood lifetime, in which 50% glycol chitosan nanoparticles could circulate for 23 h, compared to free Cy5.5 (Supporting Information Figure SI1). Next, we investigated whether optical/MR dual imaging could be used to detect tumor tissues in live animals. In SCC7 tumorbearing mice (8-10 mm in diameter), both NIRF optical and MR imaging readily detected the inoculated tumor tissues after 1 day postinjection of Cy5.5-CNP-Gd(III) (5 mg/kg). First of all, MR imaging showed tumor tissues. In particular, coronal T1-weight MR images showed a high spatial resolution of tumor tissues after 1 day postinjection (Figure 4a). The clear margin of tumor tissue in soft tissue could be completely delineated by the bright T1-weight MR image, indicating the excellent nanoparticle accumulation at the tumor site. The relative signal enhancement (RSE, %) was calculated in the region of interests (ROIs) of the T1-weighted images. After 1 day postinjection of Cy5.5-CNP-Gd(III), the ROI value at tumor strongly increased from 70 to 162, compared to that of muscle (65 to 95). Noticeable brightening appeared at tumor with the RSE value of 231%, indicating the higher tumor targeting ability of glycol chitosan nanoparticles. However, the sensitivity was not higher compared to that of optical imaging. As we expected, NIRF imaging showed a strong and sensitive fluorescence image under explore Optix system, indicating high sensitivity of NIRF imaging, but the high-resolution histological and spatial image

This work was financially supported by the Real-Time Molecular Imaging Project, 2009K 001594, Seoul R&BD Program (10524), Pioneer Research Program (2009-0081523), and Global Research Laboratory Project of MEST and by a grant from the Intramural Research Program of the KIST, and by a grant (A062254) from the Korea Health 21 R&D Project. Supporting Information Available: Experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org.

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