Facile Method To Radiolabel Glycol Chitosan Nanoparticles with

Apr 15, 2013 - Facile Method To Radiolabel Glycol Chitosan Nanoparticles with 64Cu via Copper-Free Click Chemistry for MicroPET Imaging. Dong-Eun ... ...
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Facile Method To Radiolabel Glycol Chitosan Nanoparticles with 64Cu via Copper-Free Click Chemistry for MicroPET Imaging Dong-Eun Lee,†,‡ Jin Hee Na,‡,§ Sangmin Lee,‡,§ Choong Mo Kang,∥ Hun Nyun Kim,∥ Seung Jin Han,‡,§ Hyunjoon Kim,‡ Yearn Seong Choe,∥ Kyung-Ho Jung,∥ Kyo Chul Lee,⊥ Kuiwon Choi,‡ Ick Chan Kwon,‡ Seo Young Jeong,§ Kyung-Han Lee,*,∥ and Kwangmeyung Kim*,‡ †

Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, 29 Geumgu-gil, Jeongeup, Jeonbuk 580-185, Republic of Korea ‡ Center for Theragnosis, Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul 136-791, Republic of Korea § Department of Life and Nanopharmaceutical Science, KyungHee University, Seoul 130-701, Republic of Korea ∥ Department of Nuclear Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea ⊥ Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences, Seoul 139-706, Republic of Korea S Supporting Information *

ABSTRACT: An efficient and straightforward method for radiolabeling nanoparticles is urgently needed to understand the in vivo biodistribution of nanoparticles. Herein, we investigated a facile and highly efficient strategy to prepare radiolabeled glycol chitosan nanoparticles with 64Cu via a strain-promoted azide−alkyne cycloaddition strategy, which is often referred to as click chemistry. First, the azide (N3) group, which allows for the preparation of radiolabeled nanoparticles by copper-free click chemistry, was incorporated to glycol chitosan nanoparticles (CNPs). Second, the strained cyclooctyne derivative, dibenzyl cyclooctyne (DBCO) conjugated with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator, was synthesized for preparing the preradiolabeled alkyne complex with 64Cu radionuclide. Following incubation with the 64Cu-radiolabeled DBCO complex (DBCO-PEG4-LysDOTA-64Cu with high specific activity, 18.5 GBq/μmol), the azide-functionalized CNPs were radiolabeled successfully with 64 Cu, with a high radiolabeling efficiency and a high radiolabeling yield (>98%). Importantly, the radiolabeling of CNPs by copper-free click chemistry was accomplished within 30 min, with great efficiency in aqueous conditions. In addition, we found that the 64Cu-radiolabeled CNPs (64Cu-CNPs) did not show any significant effect on the physicochemical properties, such as size, zeta potential, or spherical morphology. After 64Cu-CNPs were intravenously administered to tumor-bearing mice, the realtime, in vivo biodistribution and tumor-targeting ability of 64Cu-CNPs were quantitatively evaluated by microPET images of tumor-bearing mice. These results demonstrate the benefit of copper-free click chemistry as a facile, preradiolabeling approach to conveniently radiolabel nanoparticles for evaluating the real-time in vivo biodistribution of nanoparticles. KEYWORDS: radiolabeling, nanoparticles, copper-free click chemistry, microPET imaging

1. INTRODUCTION

excellent sensitivity and yields quantitative information from whole body PET images.7−15 To date, various radiolabeling strategies for nanoparticles have been exploited to achieve high radiolabeling yields and specific activities for obtaining high quality images by microPET.9,13−15 These include “post-conjugation of preradiolabeled chelating agent to the nanoparticles” (preradiolabeling) and “pre-conjugation of chelating agent to the nano-

Various nanoparticles have received much attention for the diagnosis and therapy of diseases.1,2 Because of their potential as nanomedicines, a great deal of effort has been made toward tracking nanoparticles in vivo to understand the organ distribution, time-dependent excretion profile, circulation time in blood, and the tumor-targeting ability.3−5 In addition, there has been research devoted to the detection of nanoparticles by the use of near-infrared fluorescence (NIRF) in vivo imaging. However, such approaches are often hampered by difficulties in accurately quantifying the NIRF signals from the specific organs in living subjects, particularly in deep tissues.1,6 Meanwhile, micropositron emission tomography (microPET) provides longitudinal imaging of the radiolabeled nanoparticles with © 2013 American Chemical Society

Special Issue: Emerging Technology in Evaluation of Nanomedicine Received: Revised: Accepted: Published: 2190

October 21, 2012 April 10, 2013 April 15, 2013 April 15, 2013 dx.doi.org/10.1021/mp300601r | Mol. Pharmaceutics 2013, 10, 2190−2198

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600 μmol, 1.5 equiv of 5β-cholanic acid) and NHS (72 mg, 600 μmol, 1.5 equiv of 5β-cholanic acid). Glycol chitosan (500 mg, 2 μmol) was dissolved in a distilled water/methanol mixture (1:1 v/v, 60 mL) and was added to 60 mL of methanol solution containing activated 5β-cholanic acid, which allowed for the formation of the amide linkage by reaction with the primary amino groups in glycol chitosan. The resulting solution was stirred for 1 day at room temperature and was then dialyzed for 3 days in a water/methanol mixture (1:3 v/v) using a cellulose membrane (MWCO 12−14 000: Spectrum Laboratories, USA). The solution was then further dialyzed for 1 d in distilled water after which it was lyophilized to give glycol chitosan-5β-cholanic acid conjugates (GC-CA). The degree of substitution (DS), defined as the number of 5β-cholanic acid molecules per one glycol chitosan polymer, was determined using a colloidal titration method, as previously described.27 The glycol chitosan conjugates were determined to contain 150 ± 4.5 molecules of 5β-cholanic acids per one glycol chitosan (glycol chitosan-5β-cholanic acid150, MW = 301 kDa). For preparation of azide (N3)-functionalized CNPs (CNPN3), azide-PEG4-NHS was chemically conjugated to the GCCA conjugate using the same method as mentioned above. Briefly, azide-PEG4-NHS (3.7 mg, 9.5 μmol) was dissolved in 500 μL of anhydrous DMSO, which was added dropwise to the conjugate solution (10 mg/mL) in anhydrous DMSO. The reaction mixture was stirred overnight at room temperature and was then dialyzed against distilled water using a cellulose membrane (MWCO 12−14 000: Spectrum Laboratories, USA) for 2 days. The reaction solution was lyophilized to give a white powder. Formation of the amide linkage between N3−PEG4 and GC-CA was confirmed by the characteristic peaks of the azide (N3) in Fourier transform infrared (FT-IR) spectra (Figure S1). Briefly, we cast thin films composed of CNP-N3 onto separate CaF2 plates at room temperature. Most of the water in the films was removed by evaporation at 40 °C in a vacuum oven for 48 h. FR-IR spectra of the dried film were observed using a 1000 PC spectroscope (PerkinElmer, USA) with an average of 64 scans at a resolution of 4 cm−1 reported. The DS of azide-PEG4 per molecule of glycol chitosan-5βcholanic acid conjugate was determined by a colloidal titration method.26 Finally, azide-functionalized CNP (CNP-N3) (1 mg/mL) in distilled water or PBS (pH 7.4) was sonicated 3 times using a probe-type sonicator (Ultrasonic Processor, GEX-600) at 90 W for 2 min, in which the pulse was turned off for 1 s after every burst of 5 s to prevent an increase in temperature. The produced CNP-N3 was well-dispersed in distilled water or PBS at 37 °C. The click reaction between CNP-N3 and DBCO derivative was evaluated by an SDS-polyacrylamide gel electrophoresis (PAGE) assay. DBCO-Cy5 (λabs 653 nm; λem 674 nm) is suitable for evaluating copper-free click chemistry with azide-functionalized molecules based on the fluorescent image. Azide-PEG4-NH2 was employed as a control group. CNP-N3 (200 μL of 2 mg/mL) was reacted with various mole ratios of DBCO-Cy5 (1−20 nmol) for 15 min at 40 °C. After 15 min of incubation, the resulting Cy5-labeled CNPs (Cy5CNPs) were loaded onto a vertical slab gel consisting of 5% stacking gel and 8% separating gel for electrophoresis. The gel was run at a constant voltage mode of 120 V in a Tris/glycine/ SDS buffer. NIRF images of the gel were obtained with a Kodak Imaging Station 4000 MM, equipped with a 150-W halogen lamp and excitation filter sets suitable for Cy5 (excitation: 653 nm, emission: 674 nm). The morphology of CNP-N3 in

particles followed by post-radiolabeling of chelating agent coupled nanoparticles with radioisotope” (postradiolabeling).13,15−17 However, these approaches have to meet a number of requirements for the successful radiolabeling of nanoparticles, for example, the reaction conditions, especially those of pH and temperature due to the sensitivity of nanoparticles and certain targeting ligands, such as antibodies and proteins attached to the nanoparticles.15,18 Furthermore, purification is generally required to reach radiochemical yields above 90% for systemic administration, especially when radiolabeling efficiency is poor under mild reaction conditions.18,19 Therefore, it would be greatly useful to develop more facile and efficient radiolabeling strategies for nanoparticles.20,21 Recently, the strain-promoted azide−alkyne cycloaddition, which is also referred to as the copper-free click reaction, has emerged as a promising functionalization method because of its high specificity, efficiency, and mild reaction conditions.19,22−24 Furthermore, various reagents, especially strained cyclooctyne derivatives, have become commercially available for the efficient conjugation of azide-functionalized biomolecules.24,25 In this regard, the approach of preradiolabeling nanoparticles through copper-free click chemistry has been considered as an alternative because of its high reactivity and mild conditions, without the need for catalysts or purification steps.19,25 Herein, we describe a facile approach to prepare radiolabeled glycol chitosan nanoparticles (CNPs) with a 64Cu via strainpromoted azide−alkyne cycloaddition strategy. In this strategy, copper-free click chemistry is employed to achieve the preradiolabeling of the chelating agent with a high radiolabeling efficiency and is followed by the postconjugation of radiolabeled chelating agent to the CNPs for radiolabeling of nanoparticles. The real-time, in vivo biodistribution and tumortargeting ability of 64Cu-CNPs were also evaluated in tumorbearing mice using a whole-body microPET imaging system.

2. EXPERIMENTAL SECTION 2.1. Materials. Glycol chitosan (Mw = 250 kDa; degree of deacetylation = 82.7%), 5β-cholanic acid, N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) were purchased from Sigma (St. Louis, MO). Anhydrous dimethyl sulfoxide (DMSO) and methanol were purchased from Merck (Darmstadt, Germany). Dibenzylcyclooctyne-PEG4-NHS ester (DBCO-PEG4-NHS) and dibenzylcyclooctyne-SETA650 (DBCO-Cy5) were purchased from Click Chemistry Tools (Scottsdale, AZ, USA). The compound, 1,4,7,10-tetraazacyclododecane-1,4,7-tris(tbutyl acetate)-10-acetic acid [DOTA-tris(t-Bu) ester], was purchased from Macrocyclics (Dallas, TX, USA). FmocLys(Dde)−OH were purchased from Anaspec (Frenont, CA, USA). 64CuCl2 was obtained from a cyclotron at Korea Institute of Radiological and Medical Sciences (KIRAMS) (Seoul, Republic of Korea). Radioactivity was measured by using an ionizing chamber (Atomlab 200, Biodex). All other chemicals were of analytical grade and were used without further purification. 2.2. Synthesis of Azide-Functionalized CNPs (CNP-N3). Self-assembled glycol chitosan nanoparticles (CNPs) were prepared by chemically conjugating hydrophobic 5β-cholanic acid (CA) to the primary amine groups of glycol chitosan polymer (GC) (Mw: 250 kDa), as described in a previous report.26 Briefly, to activate the carboxylic acid group of 5βcholanic acid with NHS, 5β-cholanic acid (150 mg, 400 μmol) was dissolved in methanol and was mixed with EDC (120 mg, 2191

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between 64Cu-DOTA-Lys-PEG4-DBCO and azide-functionalized CNP. Briefly, 64Cu-DOTA-Lys-PEG4-DBCO (0.5 mCi/ 10−9 mol of DOTA-Lys-PEG4-DBCO in 100 μL) was incubated with CNP-N3 (2 mg/mL, 400 μg in 200 μL) for 30 min at 40 °C in aqueous conditions in order to prepare radiolabeled nanoparticles. After the reaction, the efficiency of click reaction and the radiochemical yield of radiolabeled 64CuCNP were determined by radio-TLC as described above for 64 Cu-DOTA-Lys-PEG4-DBCO. The radiolabeling yield (%) for 64 Cu-CNP was determined from radio-TLC analysis as follows: (the radioactivity of 64Cu-CNP)/(the total activity) × 100. A 1 μL aliquot of the reaction mixture was spotted onto the radioTLC sheet and developed with 10% ammonium acetate/ methanol (1:1, v/v) as the mobile phase. In the 10% ammonium acetate/methanol (1:1, v/v) as the mobile phase, radiolabeled CNP with 64Cu remains at Rf 0.0, and 64CuDOTA-Lys-PEG4-DBCO migrates at Rf 0.36, respectively. The in vitro stability study was performed by incubation of 64 Cu-CNP in aqueous solution containing 50% (v/v) of mouse serum at 37 °C for 48 h. After incubation, an aliquot (100 μL) was removed at each time point (0, 2, 6, 24, and 48 h) and was measured for radioactivity using an ionizing chamber (Atomlab 200, Biodex). To verify the stability of radiolabeling, each aliquot was incubated with 10 μL of 0.1 M EDTA for 10 min and was then centrifuged through the filter tube (Millipore, MWCO 10 000, Billerica, MA) at 15 000 rpm for 30 min to separate dissociated 64Cu-EDTA from the radiolabeled CNP. The remaining radioactivity on the filter was measured and divided by the initial activity in order to determine the stability of 64Cu-CNP. 2.6. In Vivo NIRF Imaging of Cy5-CNPs in Flank Tumor-Bearing Mice. All experiments with live animals were performed in compliance with the relevant regulations and institutional guidelines of the Korea Institute of Science and Technology (KIST), and the institutional committee approved the experiments. For the evaluation of in vivo tumor targeting ability of Cy5-CNPs, squamous cell carcinoma (SCC7) tumor cells (1.0 × 106 cells) were injected subcutaneously to the flanks of 6-week-old male athymic nude mice (Institute of Medical Science, Tokyo, Japan). When the tumor reached about 100 mm2, Cy5-CNPs (10 mg/kg) was intravenously injected into SCC7 tumor-bearing mice. The time-dependent biodistribution of these materials were imaged by the eXplore Optix system (ART Advanced Research Technologies Inc., Montreal, Canada).26 Laser power and count time settings were optimized at 25 μW and 0.3 s per point. Excitation and emission spots were scanned in 1 mm steps over the selected region of interest, to generate emission wavelength scans. A 653 nm pulsed laser diode was used to excite Cy5 molecules. NIRF emission at 674 nm was collected and detected with a fast photomultiplier tube (Hamamatsu, Japan) and a timecorrelated single photon counting system (Becker and Hickl GmbH, Berlin, Germany). After intravenous injection of samples, the major organs and tumors (n = 3 per each group) were dissected from mice at predetermined time points (2, 6, 12, 24, and 48 h). NIRF images of dissected organs and tumors were obtained with Kodak Image Station 4000 MM.26 Identical illumination settings (lamp voltage, filter, exposure time, and other parameters) were used for all animal imaging experiments. The biodistribution of the injected materials for each organ were evaluated by multiplying the average NIRF intensity with the real weight of the organ, which compensates

distilled water (1 mg/mL) was observed using transmission electron microscopy (TEM, CM30 electron microscope, Philips, CA). TEM images were obtained at an acceleration voltage of 80 kV. A sample solution was placed onto a 300mesh copper grid coated with carbon. After 2 min, the dried grid was tapped with filter paper to remove distilled water and was then air-dried. A droplet of 2% (w/v) uranyl acetate was applied as negative staining. 2.3. Synthesis of DBCO-PEG4-Lys-DOTA. A bifunctional chelator agent containing the strained cyclooctyne, DBCOPEG4-Lys-DOTA, was synthesized by applying standard solid phase Fmoc strategy in Peptron Inc. (Daejeon, Republic of Korea). Briefly, as shown in Figure S2, resin conjugated FmocLys(Dde)-OH (Figure S2A) was used as a solid support for the reaction. The Fmoc group of Fmoc-Lys(Dde)-O-Resin was removed and was followed by Boc protection. The Dde group was removed with 2% hydrazine in DMF. Then, DOTA-tris(tBu ester) was then coupled with (Boc)-Lys(H)-O-Resin (Figure S2B). After coupling, the resulting (Figure S2C) was cleaved from the support by treatment with 95% trifluoroacetic acid (TFA) containing 2.5% triisopropylsilane (TIS) and 2.5% deionized water (TFA:TIS:DW = 95:2.5:2.5). Then, the DBCO with PEG4 linker (DBCO-PEG4-NHS) (Figure S2D) was coupled with DOTA-Lys to produce DBCO-Lys-PEG4DOTA (Figure S2E). The crude DBCO-Lys-PEG4-DOTA (e) product was purified and was analyzed by Shimadzu HPLC using a capcell pak C18 column, mobile phase with 0.1% TFA/ water (A) and 0.1% TFA/acetonitrile (B), and gradient conditions of 0−10% B in 2 min, 10−40% B in 10 min, and 40−70% B in 2 min at a flow rate of 1 mL/min. Under these conditions, DBCO-Lys-PEG4-DOTA (E) was typically eluted at 7.06 min. The purified DBCO-Lys-PEG4-DOTA (E) was analyzed by LC/MS (HP 1100). A major ion peak for LC/MS was found at M+ (m/z): 1111 for DBCO-Lys-PEG4-DOTA (calculated: 1111.27) (Figure S3). 2.4. Preradiolabeling: 64Cu-DOTA-Lys-PEG4-DBCO Complex. The DBCO-PEG4-Lys-DOTA was dissolved in distilled water to give a concentration of 10−3 M (10−6 mol/ mL). Cyclotron-produced 64CuCl2 solution (stock solution: 5 mCi in 50 μL of 0.01N HCl) was mixed with different concentrations of the DBCO-PEG4-Lys-DOTA (10−8 mol or 10−9 mol) in 100 μL of 50 mM sodium acetate buffer (pH 5.5), resulting in a final concentration of 5 mCi/mL (0.5 mCi/10−8 mol or 10−9 mol of DOTA-Lys-PEG4-DBCO in 100 μL), and the reaction was allowed to stand for 30 min at 40 °C. The reaction solution was then analyzed by radio-thin layer chromatography (radio-TLC) on aluminum-backed silica gel sheets (silica gel 60 F254, EMD, NJ) developed with 0.1 M citrate and 10% ammonium acetate/methanol (1:1, v/v) as the mobile phase, in order to check the radiolabeling efficiency (%). Radioactivity on radio-TLC was scanned using a system 200 imaging scanner (Bioscan, Washington, DC, USA). The retention factor (Rf) value, the distance moved by a compound with a solvent in chromatography, was calculated as follows: (distance traveled by the compound)/(distance traveled by the solvent front). In the citrate as a mobile phase, free 64Cu migrates at Rf 0.59, and 64Cu-DOTA-Lys-PEG4-DBCO remains at Rf 0.0. In the 10% ammonium acetate/methanol (1:1, v/v) as a mobile phase, free 64Cu remains at Rf 0.0, and 64CuDOTA-Lys-PEG4-DBCO migrates at Rf 0.36, respectively. 2.5. Postconjugation: Radiolabeling of CNP-N3 with 64 Cu-DOTA-Lys-PEG4-DBCO. Radiolabeling of CNPs was accomplished with the simple copper-free click reaction 2192

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for the two-dimensional accumulation image of nanoparticles in differently sized organs. 2.7. MicroPET Imaging: Biodistribution and Pharmacokinetics of 64Cu-CNPs. All experiments with live animals were performed in compliance with the relevant regulations and institutional guidelines of Samsung Medical Center, and institutional committees approved the experiments. For the in vivo microPET imaging, the SCC7 tumor reached approximately 100 mm2, radiolabeled 64Cu-CNPs were intravenously injected into SCC7 tumor-bearing mice. The stock solution of the 64Cu-CNPs used in the microPET imaging was prepared with an activity of 37 MBq/mg of 64Cu-CNPs (1 mCi/mg of 64 Cu-CNP in 1 mL saline). This 64Cu- CNPs can be used directly for the systemic administration of radiolabeled CNPs (0.2 mCi of 64Cu per 200 μg of CNPs, injection dose: 10 mg/ kg of radiolabeled CNPs). Mice were anesthetized with 1.5 to 2% isoflurane and were then placed in the camera bed in the prone position. Static PET scans of the mice were acquired at 2, 6, 24, 30, 42, and 50 h post injection, using the Inveon microPET/CT scanner, which has a 10 cm transaxial and 12.7 cm axial field of view, and operates exclusively in 3D mode (Siemens Medical Solutions, Malvern, PA, USA).28 The static microPET images were reconstructed using a 3D-ordered subset expectation maximization. The images were then processed using Siemens Inveon Research Workplace 4.0 (IRW 4.0). For each scan, the regions of interest (ROI) were drawn over tumors and major organs (liver, kidney, cardiac pool, and muscle) on whole-body axial images. All ROI values were decay-corrected, and the percent injected dose (% ID) and percent injected dose per gram of organ (%ID/g of organ) were determined by dividing the decay corrected ROI value for a given organ by the injected dose activity, with the appropriate decay corrections. ROI values within the cardiac pools for all time points, and the resulting monoexponential curve (determined using Origin software) was used to determine the blood half-life for 64Cu-CNPs. All mean values are given as ± SD. For biodistribution studies, the animals were sacrificed at 48 h postinjection. Blood and major organs (liver, intestine, kidney, spleen, heart, lung, and stomach) were subsequently dissected and counted in a Wallac 1470 Wizard automated gamma counter (PerkinElmer Life Sciences) to determine radioactivity. Percent injected dose (%ID) and percent injected dose per gram of organ (%ID/g organ) were calculated from standard control activity with appropriate decay corrections. All mean values are given as ± SD (n = 7).

Figure 1. Preparation of azide-functionalized CNPs (CNP-N3). (A) Chemical structure of azide-functionalized glycol chitosan-5β-cholanic acid conjugate (CNP-N3). (B) Schematic illustration for the fluorescence labeled CNPs with DBCO-Cy5 via copper-free click chemistry. (C) Analysis of click reaction between CNP-N3 and DBCO-Cy5 confirmed by SDS-PAGE analysis. Linear PEG4-N3 was employed as a control. (D) Average hydrodynamic diameter (298.78 ± 34 nm) of Cy5-CNPs measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM) image of CNP-N3 (scale bar = 300 nm).

appearing at approximately 2110 cm−1 in FT-IR spectra (see Supporting Information, Figure S1). Under optimal condition, the amounts of conjugated azide groups were 5 wt %, and the azide content in the CNP-N3 was 47 in each CNP, as determined by a colloidal titration method.27 The zeta potential of CNP-N3 were of 17.54 ± 0.32 mV in deionized water, indicating that CNP-N3 has a surface shell of hydrophilic GC, with a cationic charge and with azide functionalization in aqueous conditions. To verify the interaction of azide-functionalized CNPs (CNP-N3) with the ring-strained alkyne derivative, dibenzyl cyclooctyne (DBCO) via copper-free click chemistry, fluorescent dye conjugated DBCO derivative, DBCO-Cy5 (λabs 653 nm; λem 674 nm) was used for monitoring the extent of the click reaction by fluorescence under SDS-PAGE analysis (Figure 1B). When the freshly prepared CNP-N3 (400 μg) was incubated with various amounts of DBCO-Cy5 at 40 °C for 15 min, we observed that CNP-N3 with DBCO-Cy5 produced stable fluorescence dye-labeled CNPs and approximately 20 nmol of DBCO-Cy5 could be quickly conjugated with CNP-N3 within 15 min. As shown in Figure 1C, Cy5labeled CNPs (Cy5-CNPs) remained at the origin of the 5% stacking gel, while linear PEG4-N3 migrated from the origin to the bottom. The fluorescent intensities of the Cy5-CNPs remained at the starting point of PAGE-gel even though the amount of DBCO-Cy5 was increased up to 20 nmol. Also, the freshly prepared Cy5-CNPs could form self-assembled nanoparticles with an average size of 298.78 ± 34 nm, as confirmed

3. RESULTS 3.1. Preparation of CNP-N3. As shown in Figure 1A, the azide (N3)-functionalized glycol chitosan nanoparticles (CNPN3) were prepared according to procedures which were slightly modified from those of our previous reports.26 The glycol chitosan (GC) polymers (Mw: 250 kDa) were conjugated with 23 wt % of hydrophobic 5β-cholanic acid (CA) in the presence of EDC and NHS to prepare the GC-CA conjugates. Under optimal conditions, the GC-CA conjugates contained approximately 150 molecules of CA per one GC polymer, and they could form self-assembled glycol chitosan nanoparticles (CNPs) under aqueous conditions.29 The N3-PEG4-NHS was readily attached to the GC-CA conjugates through amide formation in the presence of EDC, resulting in the azidefunctionalized CNPs (CNP-N3) (Figure 1A). The formation of the amide linkage between N3-PEG4 and GC-CA was confirmed by the characteristic peaks for the azide (N3) 2193

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by dynamic light scattering (DLS) (Figure 1D). TEM images also showed that the Cy5-CNPs constructed from CNP-N3 with DBCO-Cy5 exhibited a spherical morphology with a uniform size distribution. 3.2. Preparation of DBCO-PEG4-Lys-DOTA. DBCOPEG4-Lys-DOTA was synthesized by a simple solid phase synthesis procedure (Figure 2A and Figure S2 in Supporting

condition to prepare radiolabeled CNPs (Step 2: postconjugation via copper-free click reaction). About >98% of radiolabeling yield for 64Cu-DOTA-Lys-PEG4-DBCO was achieved with a high specific activity (18.5 GBq/μmol, 0.5 Ci/μmol). There was a negligible amount ( 100) was also attempted in an effort to increase the amount of DOTA located on the surface, but the resulting conjugates suffered from poor solubility and underwent aggregation during the dialysis.30 The in vivo biodistribution and preferential tumor accumulation profiles of azide-functionalized CNPs were investigated using noninvasive NIRF imaging with Cy5-CNPs (Figure 4). As expected, Cy5-CNPs preferentially accumulated in tumor tissues, and the fluorescent signal in tumor regions gradually increased as a function of time until 48 h postinjection, indicating the excellent tumor targeting ability of Cy5-CNPs. Since the information obtained from whole-body NIRF imaging is insufficient for the accurate quantification of in vivo biodistribution and tumor-targeting efficacy, after NIRF optical imaging, animals must be sacrificed during the course of the experiment to analyze ex vivo NIRF images of major organs (Figure 4B).7 However, there are certain differences between in vivo NIRF images and ex vivo images, which are partly contributed to the poor tissue penetration depth of fluorescence and to the statistical variations among groups of animals which were sacrificed at different times. As shown in Figure 4, in particular, we found that the NIRF signal intensities of both liver and kidney in the ex vivo images were stronger than those from the NIRF whole-body images. In this regard, highly sensitive and quantitative information was obtained from in vivo microPET images, making it an attractive option for a precise and accurate evaluation of the pharmacokinetics and tumor-targeting efficacy of nanoparticles.8 As shown in Figure 5B, when 64Cu-CNPs (0.25 mCi, 200 μg) were administered to tumor-bearing mice (20 g mice), radioactivity in the tumor region gradually increased as a function of time, which reached a plateau at 50 h postinjection (6.1 ± 1.3%ID/g at 2 h postinjection and 11.3 ± 1.25%ID/g at 50 h postinjection, respectively). In general, when the radiolabeled nanoparticles are systemically injected, they are usually taken up by the

4. DISCUSSION The preparation of radiolabeled nanoparticles often must be achieved within a restricted time, mainly due to the half-life of the radionuclides which are used, and a high radiochemical yield is more favored to avoid further purification procedures.13,19 Therefore, it is ideal to efficiently radiolabel predefined nanoparticles with radionuclides immediately prior to use by simple and fast preparation. In the present study, we demonstrated a facile approach to prepare radiolabeled polymeric nanoparticles with 64Cu by a strain-promoted, azide−alkyne cycloaddition strategy, which is often referred as click chemistry.23−25 Glycol chitosan nanoparticles (CNPs) are well-suited as a model system for the preparation of radiolabeled polymeric nanoparticles by copper-free click chemistry, because CNPs spontaneously formed spherical nanoparticles in aqueous conditions and exhibited excellent properties for tumor targeting.26,29 In addition, CNPs showed a substantial biocompatibility to allow for the administration even at a higher dose (200 mg/kg) for tumor treatment, as we have reported previously.32,33 Functionalization of CNPs with azide has no significant effect on the physicochemical properties of the original CNPs, in terms of size, zeta potential, or spherical morphology. The zeta potential of azide functionalized CNPs was 17.54 ± 0.32 mV, implying that the amphiphilic GC-CA conjugate could form nanoparticles with a hydrophilic GC surface with azide functionalization. These results could be attributed to the fact that the small amounts of conjugated azide groups (5 wt %) on the GC polymer did not affect the physicochemical properties of CNPs due to the minimized consumption of amine groups on the GC polymers. As shown in Figure 1C, the CNP-N3 can react with DBCO-Cy5 to efficiently produce Cy5-labeled CNPs within 15 min, with great efficiency in aqueous conditions, even though the amount of Cy5-DBCO was increased up to 20 nmol, demonstrating the efficient and site-specific reaction between azide and DBCO. Therefore, it is anticipated that the stable triazole linker between the azide on the CNP and DBCO makes it possible to efficiently radiolabel nanoparticles with 64Cu. Moreover, this result indicates that the specific activity and radiolabeling yield of radiolabeled nanoparticles can be artificially controlled by adjusting the amount of radiolabeled DBCO complex by copper-free click chemistry.23,25 As expected, we have achieved radiolabeling of CNP-N3 with 64Cu-DOTA-Lys-PEG4-DBCO (specific activity; 18.5 GBq/μmol, 0.5 Ci/μmol), by using simple stirring of mixtures in mild conditions (for 30 min at 40 °C in aqueous solution), by copper-free click reaction (Figure 2). As a result, this straightforward method provides radiolabeling yields of over 98% and sufficiently high radioactivity without the need for the further purification (1.25 mCi/mg of CNPs in 1 mL saline; Figure 3B). Theoretically, it is anticipated that the specific activity of radiolabeled CNPs could increase by 2196

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activity using copper-click chemistry. The stable triazole linker between the azide on the CNPs and DBCO make it possible to radiolabel nanoparticles with 64Cu, efficiently and with a highradiolabeling yield. The quantitative analysis for the 64Curadiolabeled CNPs based on the microPET image demonstrated that the 64Cu-radiolabeled CNPs were accumulated effectively into tumor tissue with a prolonged circulation time in blood circulation. These results suggest that the radiolabeling of nanoparticles using copper-free click chemistry has potential as a facile and straightforward method for evaluating in vivo biodistribution and microPET analysis.

reticuloendothelial system (RES) and removed through the liver and spleen.3,11,15 As shown in Figure 5, the high radioactivity in the liver and the kidney was observed at all time points, and the retention of radioactivity in both organs was slowly reduced over time. The high radioactivity in the abdomen makes it difficult to delineate the tumor region at early time points, which often results in misleading outcomes for the initial biodistribution from whole body microPET images. Especially, when nanoparticles with longer circulation periods in the blood are employed, tumor-specific imaging is often hampered by the high background signals derived from nonspecifically distributed radioactivity.3,11,15 Therefore, at early time points (at 2 and 6 h postinjection), drawing ROIs over tumor regions on the static PET images should be carefully considered. Meanwhile, blood circulation of CNP assessed from the radioactivity in the cardiac pool was prominent at early time points but decreased gradually as a function of time. As a result, the tumor/blood ratios increased from 0.26 at 2 h postinjection, to 1.54 at 50 h postinjection, indicating a preferential accumulation of CNPs in tumor regions, mainly due to the EPR effect. MicroPET provides longitudinal whole body images with excellent sensitivity and quantitative information (Figures 4A and 5A). From the whole body microPET images, the blood half-life for the 64Cu-CNPs can be estimated by monitoring radioactivity obtained from the cardiac pool of each experimental tumor-bearing mouse over time. The blood half-life for 64Cu-CNPs was estimated to be approximately 27 h (T1/2 = 27 h). This result is very similar with our observation for the blood half-life of the fluorescent dye-labeled CNP (T1/2 = 20 h), in which the fluorescence intensities of the blood samples over time were compared among different groups of animals to obtain semiquantitative data for the blood circulation of CNP.29 Copper-free click chemistry has emerged as a new paradigm in click chemistry, promising to provide synthetic tools for the preparation of novel materials for the construction of multifunctional nanoparticles, as well as for an efficient bioconjugation method.24,34 When macromolecular precursors, such as amphiphilic block copolymers, or lipids to fabricate polymeric nanoparticles or liposomes, are grafted with azide to form a predefined nanostructure, they allow a rapid building azide-functionalized nanostructure with optimized physicochemical properties. Thus, copper-free click chemistry could provide a facile method of radiolabeling nanoparticles.19 In the case of inorganic nanoparticles (e.g., magnetic- and gold nanoparticles), the surface of inorganic nanoparticles can be functionalized with various types of azide-conjugated ligands (e.g., surfactants, polymers, and biomolecules) to enhance the chemical stability in solution. With this preparation, azideconjugated polymers could provide a means of labeling nanoparticles with various radionuclides. Moreover, this strategy could expand the functionalization of nanoparticles with various biomolecules, such as peptide ligands or antibodies for active targeting under biocompatible conditions.23,24,34

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ASSOCIATED CONTENT

S Supporting Information *

FT-IR spectra of azide-functionalized glycol chitosan nanoparticles and synthetic scheme for DBCO-PEG4-Lys-DOTA using solid-phase Fmoc strategy. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*K.-H.L.: Tel.: +82-2-3410-2630; fax: +82-2-3410-2639; e-mail: [email protected]. K.K.: Tel.: +82-2-958-5916; fax: +822-958-5909; e-mail: [email protected]. Author Contributions

D.-E.L. and J.H.N. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the KIST intramural project (2E24051), Translational Research Program (TRP), the Global Research Laboratory (GRL), the Fusion Technology Project (2010-50201), and the MD-PhD Program (2010-0019863).

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REFERENCES

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