Article pubs.acs.org/journal/abseba
Polymeric Micelle Platform for Multimodal Tomographic Imaging to Detect Scirrhous Gastric Cancer Yutaka Miura,†,‡ Atsushi B. Tsuji,†,§ Aya Sugyo,§ Hitomi Sudo,§ Ichio Aoki,§ Masayuki Inubushi,§ Masakazu Yashiro,⊥ Kosei Hirakawa,⊥ Horacio Cabral,|| Nobuhiro Nishiyama,# Tsuneo Saga,*,§ and Kazunori Kataoka*,‡,||,△ ‡
Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan § Diagnostic Imaging Program, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan ⊥ Department of Surgical Oncology, Osaka City University, Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan || Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan # Polymer Chemistry Division, Chemical Resources Laboratory, Tokyo Institute of Technology, R1-11, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan △ Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan S Supporting Information *
ABSTRACT: Scirrhous gastric cancer (SGC) is a recalcitrant tumor, which is among the most lethal cancers. A critical issue for the improvement of SGC prognosis is the lack of an effective imaging method for accurate detection and diagnosis. Because combined nuclear medicine imaging with magnetic resonance imaging (MRI) has the ability to detect cancer with high sensitivity, and quantitation and spatial resolution, it has potential to overcome the issues with SGC detection. Herein, we designed and synthesized a new block copolymer poly(ethylene glycol)-b-poly(γ-benzyl L-glutamate) linked with a chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-PEG-b-PBLG) to provide a platform for multimodal tomographic imaging. We then successfully prepared DOTA-functionalized polymeric micelles (DOTA/m) measuring 30 nm in diameter, which is an appropriate size to penetrate deeply into tumors with thick fibrosis, including SGC. 111In-labeled DOTA/m highly accumulated in Colon-26 tumors (mouse colon cancer with hyperpermeability), but also in OCUM-2 M LN tumors (SGC with hypopermeability), clearly depicting both tumors by single photon emission computed tomography (SPECT). Gd-labeled DOTA/m clearly visualized OCUM-2 M LN tumors by MRI with high spatial resolution. Moreover, 111In/Gdlabeled micelles, as well as the mixture of 111In- and Gd-labeled DOTA/m demonstrated the capability of this system for selective multimodal SPECT/MR imaging of SCG. Our findings support 111In/Gd-DOTA-labeled micelles as a clinical translationable modality for multimodal tomographic imaging capable of detecting SGC. KEYWORDS: polymeric micelles, in vivo imaging, single photon emission computed tomography, magnetic resonance imaging, multimodality imaging poor, and the 5-year survival rate is 10−15%.1,3,4 Therefore, there is a pressing need to develop new and effective treatments for SGC. To efficiently develop new effective therapy, noninvasive imaging with high sensitivity for precisely monitoring the efficacy of treatments in both SGC patients and animal models is necessary.
1. INTRODUCTION Scirrhous gastric cancer (SGC) is a highly intractable gastric carcinoma and accounts for approximately 10% of all gastric carcinomas.1,2 Signet ring cell carcinoma or poorly differentiated carcinoma diffusely infiltrates the stomach wall and induces reactive fibrosis in patients with SGC, resulting in fibrous-like thickening of the gastric wall.1 The common features of SGC include rapidly progressive invasion and a high frequency of dissemination to the peritoneum.1 SGC prognosis is extremely © XXXX American Chemical Society
Received: March 20, 2015 Accepted: September 21, 2015
A
DOI: 10.1021/acsbiomaterials.5b00142 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Inc., Dallas, TX), sodium cyanoborohydride (NaBH3CN, Tokyo Chemical Industry, Cp., Ltd., Tokyo, Japan), N,N-dimethylacetamide (DMAc, 98%) and other reagents were used without further purification. Dulbecco’s Modified Eagle medium (DMEM) and fetal bovine serum were purchased from Sigma (St. Louis, MO). 111In-chloride was purchased from Nihon Medi-Physics (Tokyo, Japan). Acetal-poly(ethylene glycol)-amine (acetalPEG-NH2, Mn, = 12000, PDIGPC = 1.03) was prepared in accordance with the previously described synthetic method.16 Gd-DOTA complex and InDOTA complexes were synthesized by previously reported method.17,18 2.2. Measurements. 1H-nuclear magnetic resonance (NMR) spectra were recorded using a JNM-ECS400 (400 MHz) instrument (JEOL, Tokyo, Japan) with DMSO-d6 containing 0.05% tetramethylsilane at 22 °C. Size exclusion chromatography (SEC) was performed at 40 °C using an HLC-8220 gel permeation chromatography system (Tosoh, Tokyo, Japan) equipped with a TSKgel G3000HHR column (Tosoh, linear, 7.8 mm × 300 mm; pore size, 7.5 nm; bead size, 5 μm; exclusion limit, 6 × 104 g/mol), a TSKgel G4000HHR column (Tosoh, linear, 7.8 mm × 300 mm; pore size, 20 nm; bead size, 5 μm; exclusion limit, 4 × 105 g/mol), and a TSKgel guard column HHR-L (Tosoh) in DMF containing lithium bromide (10 mM) at a flow rate of 0.5 mL/min. The number-average molecular weight (Mn) and PDI (Mw/Mn) of PEG derivatives were calculated on the basis of the PEG calibration. The Mn and degree of polymerization of amino acid were calculated using 1H NMR spectra. Polymerization of the N-carboxy anhydride of γ-benzyl L-glutamate was monitored using an infrared spectrometer with an IR report-100 (JASCO, Tokyo, Japan) and an NaCl plate. The sizes of the micelles were measured by dynamic light scattering (DLS) using two types of zetasizers (Malvern Zetasizer Nano ZS90) equipped with a 4.0 mW He−Ne laser operating at 633 nm, or a 50.0 mW DPSS laser operating at 532 nm with 90° collecting optics. Data were analyzed using Malvern Dispersion Technology 4.20. Gadolinium concentration in the micelles was determined by inductively coupled plasma mass spectrometry (ICP-MS) using a 7700x ICP-MS (Agilent Technologies, Santa Clara, CA; RF power, 1550 W; sampling depth, 8.0 mm; plasma gas current, 16 L/min; carrier gas flow rate, 1.02 L/min; peristaltic pump, 0.10 rps; monitoring mass, m/z 157 and 158 (Gd); integration interval, 0.1 s; sampling period, 0.31 s). 111In-labeled DOTA/m was analyzed by using a high-performance liquid chromatography (HPLC) system (370 PUMP and UV/vis 157, Gilson, Middleton, WI) with an RI detector (Gabi, Raytest, Straubenhardt, Germany) equipped with a Superdex 200 column (GE Healthcare, Little Chalfont, UK, linear, 10 mm × 300 mm; bead size, 13 μm; exclusion limit, 1.3 × 106 g/mol) and an isocratic mobile phase of 10 mM phosphate buffer with 150 mM NaCl (pH 7.4) at a flow rate of 0.5 mL/min. 2.3. Cells and Animals. The murine colon carcinoma cell line Colon-26 was purchased from the RIKEN BioResource Center (Tsukuba, Japan). The OCUM-2 M LN cell line was established from a human diffuse-type SGC.19 The cells were maintained in DMEM with 5% fetal bovine serum in a humidified incubator maintained at 37 °C with 5% CO2. BALB/c-nu/nu male mice (5 weeks old; Japan SLC, Hamamatsu, Japan) were maintained under specific pathogen-free conditions. The mice were inoculated subcutaneously with Colon-26 (1 × 106 cells/mouse) or OCUM-2 M LN (3 × 106 cells/mouse) cells under isoflurane (Mylan, Tokyo, Japan) anesthesia. For orthotopic SGC model mouse, OCUM-2 M LN cells (1 × 107 cells/mouse) were transplanted subserosally into the gastric walls following previously described method.20 The animal experimental protocol was approved by the Animal Care and Use Committee of the National Institute of Radiological Sciences, and all animal experiments were conducted in accordance with the institutional guidelines regarding animal care and handling. 2.4. Synthesis of Acetal-PEG-b-PBLG. Acetal-poly(ethylene glycol)-b-poly(γ-benzyl L -glutamate)-propionate (Acetal-PEG-bPBLG) was synthesized in accordance with the previously described synthetic method with a minor modification.21 The N-carboxyanhydride of γ-benzyl-L-glutamate (1.32 g, 5.01 mmol) in DMF (30 mL) was added to acetal-PEG-NH2 in DMF (20 mL) and stirred at 25 °C for 3 days in an argon atmosphere. Polymerization was terminated by adding N-succinimidyl propionate (1.32 g, 5.01 mmol). After stirring at room temperature for 6 h, the reaction mixture was transferred to a
Nuclear medicine imaging, such as positron emission tomography (PET) and single photon emission computed tomography (SPECT), is noninvasive diagnostic imaging method with high sensitive and quantitative properties. In most cases, 18 F-fluorodeoxyglcuose (FDG) PET is widely used in clinics,5 as it provides clear visualization of solid tumors, which is closely correlated with the expression of the glucose transporter GLUT-1. However, GLUT-1 is not overexpressed in signet ring cell carcinoma and nonsolid type poorly differentiated carcinoma.6 Therefore, imaging of SGC has not been achieved by using 18F-FDG.6−8 Furthermore, nuclear medicine imaging has the limitation for spatial resolution (several mm), resulting difficulty of accurate detection for the precise position of cancer. In contrast, magnetic resonance imaging (MRI) has high spatial resolution ( 99%) was purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan) and distilled over CaH2 before use. N-carboxy anhydride of γ-benzyl L-glutamate (Chuo Kaseihin Co., Inc., Tokyo, Japan), N-succinimidyl propionate (Wako Pure Chemical), p-NH2−Bn-DOTA (Macrocyclics, B
DOI: 10.1021/acsbiomaterials.5b00142 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
2.10. Biodistribution of In/Gd-DOTA/m and 111In-DOTA/m. To evaluate the plasma clearance and tumor accumulation by ICP-MS, we intravenously injected mice (18−20 g body weight) with In/GdDOTA/m, free Gd-DOTA, and In-DOTA at a dose of 100 μg per mouse on a In or Gd basis. The mice were sacrificed at 1, 4, 8, 24, 48, and 96 h after injection (n = 5). Blood was collected from the inferior vena cava, heparinized, and centrifuged to obtain the plasma. The tumors were excised, washed with PBS, and their weights were measured by electronic balance. All samples were mixed with conc. HNO3, heated to dryness, and redissolved in 1% HNO3 (1.0 mL). The In and Gd concentration were then measured by ICP-MS. For the measurement of organ distribution by radioactivity, mice (20.5 ± 1.6 g body weight) were intravenously injected with 37 kBq of 111In-DOTA/m (250 μg polymer, 10−20 mL/kg), when subcutaneous tumors reached a diameter of approximately 10 mm. Group of mice were euthanized at 1, 4, 8, 24, 48, and 96 h after injection. Tumors and major organs were removed and weighed (n = 5 for each time point), and radioactivity counts were measured using a gamma counter (Aloka, Tokyo, Japan). Data are expressed as the decay-corrected percentage of injected dose per gram of tissue (%ID/g). 2.11. SPECT/CT Imaging with 111In-DOTA/m, 111In/Gd-DOTA/m, and a Mixture of 111In-DOTA/m and Gd-DOTA/m. A 1.85 MBq aliquot of 111In-labeled micelles (450 μg polymer, 10 mL/kg), such as 111 In-DOTA/m, 111In/Gd-DOTA/m, or a mixture of 111In-DOTA/m and Gd-DOTA/m (1/1 = v/v, 20 mL/kg) was intravenously injected into a mouse bearing Colon-26 or OCUM-2 M LN tumor. At 4, 24, 48, 72, and 96 h after injection, the mice were anesthetized by isoflurane inhalation and imaged using the VECTor/CT SPECT/CT pre-clinical imaging system with a multipinhole collimator (MILabs, Utrecht, The Netherlands). SPECT data were acquired for 15 min at 4 h, 20 min at 24 h, 25 min at 48 h, 30 min at 72 h, and 35 min at 96 h after injection. SPECT images were reconstructed using a pixel-based ordered-subsets expectation maximization algorithm with 8 subsets and 2 iterations on a 0.8 mm voxel grid without attenuation correction. CT data were acquired with the X-ray source set at 60 kVp and 615 μA and images were reconstructed using a filtered back-projection algorithm for cone beam. 2.12. Autoradiography with 111In-DOTA/m. BALB/c-nu/nu mice bearing orthotopic OCUM-2 M LN tumor were intravenously injected with 37 kBq of 111In-DOTA/m (250 μg polymer, 20 mL/kg). Forty-8 h after administration, the stomach including the orthotopic tumor was excised, and washed with PBS and quickly frozen in an optimal-cutting-temperature compound (Sakura Finetek Japan, Tokyo, Japan). Paraformaldehyde-fixed sections (20-μm thick) were exposed to an imaging plate (Fuji Film, Tokyo, Japan) at room temperature. One day later, the imaging plate was read using an FLA-7000 imaging plate reader (Fuji Film), and then the sections were stained with hematoxylin and eosin (H&E). 2.13. MR Imaging with Gd-DOTA/m and In/Gd-DOTA/m. Twodimensional, multislice, T1-weighted images were acquired before the administration of micelles, and 0.5, 1, 24, 48, and 72 h after injection of Gd-DOTA/m or In/Gd-DOTA/m at a dosage of 0.0075 mmol/kg based on Gd, by using a conventional spin−echo sequence with a preclinical MRI scanner (ICON, Bruker Biospin, Ettlingen, Germany) with solenoid mouse body coil (Bruker Biospin). Also, to eliminate fat signal, an inversion−recovery prepared “fat” image was obtained with the same slice orientation before Gd-DOTA/m injection using rapid acquisition with relaxation enhancement (RARE) sequence. GdDOTA/m or In/Gd-DOTA/m was intravenously administered to the OCUM-2 M LN and Colon-26 tumor mouse model. The mice were anesthetized with isoflurane during MRI, and rectal temperatures were monitored and maintained at approximately 37 °C with warm water. The imaging parameters for T1-weighted images were as follows: TR/TE = 400/10.6 ms; FOV, 40.0 × 40.0 mm2; matrix, 256 × 256; resolution, 156 μm × 156 μm; number of slices, 11; slice thickness, 1.0 mm; slice gap, 0.5 mm; slice direction, transaxial; and NEX, 12. The imaging parameters of “fat” image were, as follows: TR/effective TE = 5000/20 ms; inversion time, 450 ms; FOV, 40.0 × 40.0 mm2; matrix, 128 × 128; spatial resolution, 312 μm × 312 μm × 1000 μm; number of slices, 9; slice thickness, 1.0 mm; slice gap, 0.5 mm; slice
cellophane tube (Spectra/Pro 6 membrane: MWCO, 3500) and dialyzed for 1 day against DMF/water and water (v/v) (the DMF/water ratio was changed gradually from 1/0 to 0/1), followed by lyophilization to yield acetal-PEG-b-PBLG: yield 87%, Mn, NMR = 20800, PDIGPC = 1.09, DPPBLG = 40. 1H NMR (400 MHz, DMSO-d6): δ (ppm) = 1.10 (t, 3H, CH3-CH2−CO−), 1.11 (t, 6H, CH3-CH2−O- acetal group), 1.72−2.70 (m, 160H, -CH2-CH2- PBLG side chain), 3.34−3.80 (m, 1090H, -CH2CH2-O-PEG backbone), 3.80−4.35 (m, 40H, -CH- PBLG backbone), 4.70−5.30 (m, 80H, -CH2-C6H5 PBLG side chain), 7.02−7.50 (m, 200H, −CH2−C6H5 PBLG side chain). 2.5. Preparation of DOTA End-Functionalized PEG-b-PBLG. To afford aldehyde end-groups on PEG-b-PBLG, an aqueous HCl solution (pH 2, 7.6 mL) was combined with acetal-PEG-b-PBLG (305 mg, 1.53 × 10−2 mmol) in DMF (5.0 mL), and then the mixture was stirred at room temperature. After 2 h of stirring, the solution was neutralized with 0.5 M NaOH solution, in a dropwise manner. Then, p-NH2−Bn-DOTA (98.2 mg, 1.53 × 10−1 mmol) was added to the reaction mixture and stirred at room temperature for overnight. The Schiff base obtained was reduced to a secondary amine using a catalytic amount of NaBH3CN (ca. 10 mg) and the mixture was stirred for another 24 h. The reaction mixture was transferred to a cellophane tube (Spectra/Pro 6 membrane: MWCO, 3500) and dialyzed for 1 day against DMF/water and water (v/v) (the DMF/water ratio was changed gradually from 1/0 to 0/1), followed by lyophilization to yield DOTAPEG-b-PBLG: yield 91%, Mn, NMR = 21500, PDIGPC = 1.07, DPPBLG = 40. 1 H NMR (400 MHz, DMSO-d6): δ (ppm) = 1.02 (t, 3H, CH3−CH2− CO−), 1.76 (m, 2H, −NH−CH2−CH2−CH2−O−, DOTA residue), 1.78−2.70 (m, 160H, −CH2−CH2− PBLG side chain), 2.80−3.80 (m, 1090H, −CH2−CH2−O−PEG backbone + m, 16H, −CH2−CH2−N−, DOTA residue), 3.80−4.35 (m, 40H, −CH− PBLG backbone), 4.70− 5.30 (m, 80H, −CH2−C6H5 PBLG side chain), 6.50 (d, J = 8.4 Hz, 2H, ArH, DOTA residue), 6.92 (d, J = 8.4 Hz, 2H, ArH, DOTA residue), 7.02−7.50 (m, 200H, −CH2−C6H5 PBLG side chain), 7.50−8.50 (br, 35H, −NH−CO−, PBLG backbone). 2.6. Preparation of DOTA/m. DOTA-PEG-b-PBLG (45 mg) was dissolved in DMAc (2.5 mL) and stirred for 1 h. DMAc was removed by a vacuum line to prepare a polymer film, and then Millipore water (10 mL) was added, in a dropwise manner. After stirring overnight, the solution was transferred to a cellophane tube (Spectra/Pro 6 membrane: MWCO, 3500) and dialyzed for 1 day against water. The micelle was characterized using a zetasizer. 2.7. Radiolabeling of DOTA/m. DOTA/m (90−120 μg/40 μL) in 0.1 M acetate buffer (pH 6.0) was incubated with 600 kBq of 111 In-acetate (10 μL) for 30 min at 37 °C. The radiochemical yield of 111In-labeled DOTA/m was determined by HPLC on a Superdex 200 column as described above. 111In-DOTA/m was purified using a Sephadex G-50 column (GE Healthcare) with 0.1 M acetate buffer (pH 6.0). Radiochemical purity was determined by HPLC. The size distribution of 111In-DOTA/m was measured by DLS. 2.8. Gd-Labeling of DOTA/m (Gd-DOTA/m). Argon was bubbled through the DOTA/m solution (25 mL), including 2.26 × 10−6 mol DOTA residue for 20 min to remove any oxygen. After adding GdCl3·6H2O (1.82 × 10−4 mol), the mixture was sonicated for 5 min at room temperature and stirred for another 3 h at 60 °C. The solution was transferred to a cellophane tube (Spectra/Pro 6 membrane: MWCO, 3500) and dialyzed for 1 day against water. The micelles were characterized using a DLS. The percentage of Gd loading was confirmed by ICP-MS and estimated to be 81%. 2.9. Preparation of Dual-Labeled DOTA/m (111In/Gd-DOTA/m). The solution of presynthesized Gd-DOTA/m was lyophilized to obtain the Gd-chelated DOTA-PEG-b-PBLG (Gd/DOTA-PEG-b-PBLG) as a white powder. The obtained Gd/DOTA PEG-b-PBLG (22.5 mg) and DOTA-PEG-b-PBLG (22.5 mg) were dissolved in DMAc (2.5 mL) and stirred for 1 h. Then, 111In/Gd-DOTA/m was prepared in the same manner described in section 2.7 and 2.8. Radiochemical purity and stability were determined by HPLC. The size distribution of 111In/GdDOTA/m was measured by DLS, and the concentration of Gd loading was confirmed by ICP-MS. For ICP-MS analysis of the biodistribution study, as well for MR imaging, cold 113In and Gd dual-labeled micelles (In/Gd-DOTA/m) was prepare in the same manner. C
DOI: 10.1021/acsbiomaterials.5b00142 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering direction, transaxial; and NEX, 2. The MRI data were reconstructed and analyzed using ParaVision software (Bruker Biospin) and analyzed using Image-J (NIH, MD, USA) and Osirix (Ver. 3.9.4, 64 bit, Pixmeo, Switzerland). To exclude the fat tissue from the T1-weighted images, following image processing was performed: (1) the “fat” image was interpolated linearly to 256 × 256 matrix; (2) binarization of the “fat” image was performed using RenyEntropy method on Image-J;22 (3) the calculated fat binary map was subtracted from the T1-weighted image; and (4) the fat excluded T1-weighted image was scaled using Rainbow_color on Osirix. 2.14. Statistical Analysis. Tumor uptake data were analyzed by two-way repeated-measures analysis of variance and Student’s t-test (two-tailed). P-values of
