Research Article | Open Access
Synthesis and In Vitro and In Vivo Evaluation of a New 68Ga-Semicarbazone Complex: Potential PET Radiopharmaceutical for Tumor Imaging
In an attempt to develop new tumor imaging radiotracers with favorable biochemical properties, we have synthesized new 68Ga-2-acetylpyridine semicarbazone (68Ga-[APSC]2) as a potential positron emission tomography (PET) tumor imaging agent using a straightforward and a one-step simple reaction. Radiochemical yield and purity were quantitative without HPLC purification. Biodistribution studies in nude mice model bearing human MDA-MB-231 cell line xenografts displayed significant tumor uptake of 68Ga-[APSC]2 radiotracer after 2 h postinjection (p.i.). The initial results demonstrate that 68Ga-[APSC]2 radiotracer may be useful probe for detecting and staging of hypoxic tumor using PET imaging modality.
The physical properties of the positron emitter gallium-68 (68Ga) and its availability from 68Ge/68Ga generator, together with the well-known coordination chemistry of gallium, make it one of the most attractive radionuclides for PET imaging. Therefore, varieties of 68Ga-labeled bioactive molecules for potential use beyond the measurement of glucose metabolism were developed and investigated [1–3]. This development will certainly help nuclear medicine centers without the on-site availability of a cyclotron and consequently patient care improvement.
Heterocyclic thiosemicarbazones, semicarbazones, and their metal complexes have been extensively investigated as potential antitumor agents [4–12]. Most studied thiosemicarbazones and semicarbazones were the pyridine-based compounds which might be attributed to their resemblance to pyridoxal metabolites that attached to coenzyme B6-dependent enzymes and caused enzyme inhibition [4, 11]. Varieties of metallic PET and single photon emission computed tomography (SPECT) radionuclides were incorporated into thiosemicarbazones chelates. Among these radionuclides, 67/68Ga and copper-62/64 (62/64Cu) have shown considerable success for targeting hypoxic and normoxic tumor xenografts in athymic mouse models [12–19].
Recently, radiolabeled 67Ga-thiosemicarbazone complex was synthesized with high radiochemical yield and purity. Biodistribution study in fibrosarcoma bearing mice revealed specific tumor accumulation after 2 h postinjection (p.i.) which may suggest that 68Ga is a better candidate for tumor imaging than 67Ga .
Due to the well-known coordination chemistry of gallium, high stability of GaIII-semicarbazone in comparison with its thiosemicarbazone complexes , and its enhanced antineoplastic activity, we were interested in developing new radiotracers with favorable biochemical properties; we here report the synthesis, characterization, and preclinical evaluation of new 68Ga-2-acetylpyridine semicarbazone (68Ga-[APSC]2) as a potential PET imaging agent for hypoxic tumor.
2.1. Materials and Measurements
The chemicals used in the study were all of analytical reagent grade, were purchased from Aldrich, and were used without further purification unless stated. Commercial 68Ga-generator based on a SnO2 phase adsorbing was obtained from iThemba Lab, South Africa. 67Ga was produced by the bombardment of enriched zinc-68 (68Zn) targets (1 g ± 10%) with 30 MeV protons from the Cyclone 30 cyclotron internal beam using the 68Zn (p, 2n) 67Ga nuclear process. Sep-Pak cartridges were purchased from Waters-Millipore. TLC-SG sheets were purchased from Grace Sciences Inc. High Performance Liquid Chromatography (HPLC) analysis was carried out on Luna, Phenomenex C-18 reversed phase column (analytical, 250 mm × 4.6 mm). The solvent system used was isocratic (eluent: ACN/H2O 95/5, with 0.1% TFA at flow rate of 1.0 mL/min). A Jasco chromatographic system equipped with a variable wavelength ultraviolet monitor and in tandem with a Canberra flow through radioactivity detector was used. Ultraviolet absorption was monitored at 254 nm. Chromatograms were acquired and analyzed using BORWIN software. Melting points were determined on a Thomas-Hoover Unimelt capillary melting point apparatus. Mass spectroscopy was run on Quattra electrospray mass spectrometer (ES-MS). Elemental analyses were performed on a Perkin Elmer CHN 2400 analyzer. IR bands were recorded on a Perkin Elmer spectrophotometer 1000 in the spectral range 200–4000 cm−1 with sample in the form of KBr pellets. 1H-13C NMR spectra were obtained in DMSO-D6 on JEOL NMR 400 MHz. Log was calculated by potentiometric methods as described in the literature .
2.2. 2-Acetylpyridine Semicarbazone (APSC)
APSC compound was synthesized using method reported previously . In brief, aqueous solution (20 mL) of semicarbazide hydrochloride (1.11 g, 1.0 mmol) was added to aqueous solution (20 mL) of 2-acetyl pyridine (1.20 g, 1.0 mmol) in the presence of sodium acetate (1.36 gm, 1.0 mmol). The reaction mixture was stirred vigorously for 2 h. The crystalline white product was filtered off and washed with ether. Yield: 96%, mp = 188–192°C. [C8H10N4O] elemental analysis (calculated): C: 54.0 (53.9%), H: 5.8 (5.7%), and N: 31.2 (31.4%). IR (): (NH: 3473); (N: 3375); (C: 2280); (C=Oas: 1686); (C=Osy: 1579); (C=N: 1443); (C–O: 1104); (heterocyclic bases: 771, 626, 561, and 448). NMR (, ppm, DMSO-d6): 3.39 (3H, C); 7.3 (2H, N); 7.3 (1H, CH=C); 7.7 (1H, CH=C); 8.3 (1H, CH–C); 8.5 (1H, CH–N); 9.5 (1H, NH). (-ppm, DMSO-d6): 12 (C); 120.7 (C–H); 123.8 (C–HC); 136 (C–H); 145 (C=N); 148 (C–HD); 155 (C–N=C); 157 (C=O). (ES) 179 ([M).
2.3. 2-Acetylpyridine Semicarbazone Ga-Complex (Ga-[APSC]2)
Ga-[APSC]2 complex was prepared according to previously published method with slight modification . To the ligand APSC (0.019 g, 0.106 mmol), dissolved in hot ethanol (EtOH, 10 mL), was added GaCl3 in EtOH solution (1 mL, 0.05 mmol) drop-wise. The reaction mixture was refluxed for 2 h under N2 atmosphere with vigorous stirring. The reaction mixture cooled down to room temperature and kept under N2 atmosphere until the yellow-white product precipitated. Precipitate was then filtered and washed with ether. Yield: 66%. M.P. = 207°C. [C16H20N8O2Cl Ga, Mr 461] elemental analysis (calculated): C: 41.3 (41.6%), H: 4.6 (4.3%), N: 24.4 (24.3%), and Cl 15.56 (15.1%). IR (): (NH, 3444); (NH2: 3245); (CH3: 2365); (C=Oas: 1678); (C=N: 1528); (C–O: 1066); (Ga-N: 540); (Ga-O: 470), 3114 (C–H). NMR (1H, ppm, DMSO-d6): 1.08 (3H, CH3); 7.3 (2H, NH2); 7.6 (1H, CH=C); 8.1 (1H, CH=C); 8.3 (1H, CH–C); 8.7 (1H, CH–N); 9.8 (1H, NH). (13C-ppm, DMSO-d6): 15 (CH3); 120.7 (CH–C); 123.8 (C=C); 136 (C=C); 146 (C=N); 148 (C–N); 157 (C–N=C); 65 (C–O). (ESI+) 427 ([M]+). Log = 22.
68Ge/68Ga generator was eluted with suprapure HCl (0.6 M, 6 mL) in 0.5 mL fractions. The two fractions with the highest 68GaCl3 (37–300 MBq) activity were generally used for labeling purposes [23, 24]. The 68Ga-[APSC]2 complex was prepared by the addition of APSC (50 μg) to a screw-cap vial containing 68GaCl3 (185–300 MBq) dissolved in 0.1% acetic acid/methanol (AcOH/MeOH, v/v; pH = 4.5). The reaction mixture was heated for 30 min at 90°C and cooled to room temperature, followed by the dilution with normal saline, and the product was passed via a 0.22 μm membrane filter to be ready for injection. The same procedure was repeated with 67Ga-[APSC]2.
2.5. Stability in Plasma
For stability in plasma, 68Ga-[APSC]2 complex (100 μL, 20 μCi each) was incubated with human plasma (500 μL) in duplicate at 37°C for 1–4 h. This was followed by precipitation using a mixture of ACN/EtOH (400 μL, 1/1 v/v) and centrifugation at 5000 rpm for 5 min. The supernatant layer was then analyzed by HPLC under the conditions described above.
2.6. In Vivo Biodistribution and Tumor Targeting
Approval for the animal protocol used in this study was obtained from the Institutional Animal Care and Use Committee. Animal biodistribution experiments were performed according to the international regulations governing the safe and humane use of laboratory animals in research . MDA-MB-231 breast cancer cell line is known to be induced by hypoxic tumor cells . Human MDA-MB-231 xenograft mouse models were used for in vivo tumor targeting experiments. For the implantation of tumor xenografts, approximately 3 × 106 MDA-MB-231 cells in suspension of 100 μL sterile saline were injected subcutaneously into the right thigh of each mouse as reported in the literature . Tumors were allowed to grow for 2-3 weeks in which tumors had reached weights of ~500 mg. Mice were injected via the lateral tail vein with 100 μL of the radiotracers formulated in saline. Each dose contained ~20 μCi (740 kBq) of the 68Ga-[APSC]2 radiotracer. Animals were sacrificed at different time intervals (4 mice at each interval) and tissues of interest were dissected, weighed, and assayed for radioactivity. The percentage of the injected dose per gram (% ID/g) was then calculated by counting all tissues in a γ-well counter using a stored sample of the injection solution as a standard to estimate the total dose injected per mouse.
2.7. Statistical Analysis
Data are expressed as mean ± S.D. where appropriate. For data comparisons, Student’s -test was performed for the mean values using Graph-Pad Software (Graph-Pad Software Inc., San Diego, CA, USA). A probability value of was considered statistically significant.
3. Results and Discussion
3.1. Organic Chemistry
The one-step synthesis of the monosemicarbazones proceeds by the reaction of 2-acetylpyridine and semicarbazides in the presence of acetic acid in aqueous media. Similarly, the reaction of APSC with GaCl3 has furnished Ga-[APSC]2 complex in good yield as shown in Scheme 1. Elemental analysis measurements for APSC ligand and Ga-[APSC]2 reference complex were found to correspond to the calculated results. Chemical purity of Ga-[APSC]2 complex was found to be greater than 99% as represented by a single peak in the HPLC chromatogram with 12.5 min retention time (Figure 1).
The IR bands in the region 3160–3440 cm−1, attributed to the symmetrical stretching mode (NH2) in the spectra of the ligand, were shifted to 3460–3210 cm−1 in those of the complex. The coordination of the ligand APSC− via the azomethine nitrogen is reflected by the shift of (C=N) band at 1443 cm−1 to higher frequency at 1528 cm−1 for Ga-[APSC]2 complex and complexation viathe C=O strong bands at 1678 cm−1 and 1579 cm−1 was attributed to (C=O) asy-, symmetrical, respectively. The (CN) band for also shifted to higher frequency at 1497 for the Ga- complex, indicating coordination of the ligand viathe pyridine nitrogen atom. Medium intensity bands at 540 and 470 cm−1 were assigned to stretching vibrations (Ga-N) and (Ga-O), respectively, whereas weak bands at 771, 626, and 561 cm−1 were assigned to heterocyclic ring.
The NMR spectra of the ligand APSC− and its Ga(III) complex were recorded in DMSO-d6. The coordination of two tridentate monoanionic ligands to Ga(III) through oxygen and azomethine nitrogen atoms resulted in upfield shifts of two quaternary carbon atoms bound to oxygen atoms from 157 ppm for APS to 65 for Ga-[APSC]2 and downfield shifts of the methyl carbons bound to the azomethine groups from 12.15 ppm for APS− to 15.16 and 14.18 ppm for the pyridine rings of Ga-[APSC]2. The carbons ortho to the nitrogen atom were shifted upfield correspondingly by 8.1 and 7.7 ppm for C1 and C2 and 7.3 and 8.5 ppm for C3 and C4, which indicates involvement of the pyridine nitrogen atoms in coordination with Ga(III).
The ES mass spectrum of Ga-[APSC]2 complex recorded in the positive mode showed a 100% relative intensity (R.I.) peak at 427 which corresponds to the theoretical isotopic distribution excluding chloride counter ion.
Due to the well-known coordination chemistry of Ga(III), high stability of Ga(III)-semicarbazone complexes, and their enhanced antineoplastic activity and in an attempt to develop new radiotracers with favorable biochemical properties for hypoxic tumor imaging, we synthesize 67/68Ga-[APSC]2 as a potential PET and SPECT tumor imaging agent. The synthetic approaches for the preparation of 68Ga-APSC chelates were simple and straightforward and gave 68Ga-[APSC]2 complexes in quantitative radiochemical yields and purities as assessed by TLC and HPLC in less than 60 min. The values for 68GaCl3 and 68Ga-[APSC]2 complex were 0.0 and 0.9, respectively (Figure 2), and the retention times for the same compounds were 4.12 and 12.70 min, respectively (Figure 3), which correspond to the reference sample (Figure 1).
3.3. Stability in Plasma
The proteolytic degradation of the 68Ga-[APSC]2 complex was determined in human plasma in vitro. The proteolytic degradation of the 68Ga-[APSC]2 complex revealed that this complex remained sufficiently stable (95%) during incubation at 37°C for at least 1 h and then started degrading to 75 and almost 50% after 2 and 4 h of incubation, respectively.
3.4. In Vivo Biodistribution and Tumor Uptake
The biodistribution data in nude mice bearing human MDA-MB-231 cell line xenografts at 1, 2, and 4 h p.i. ( for each time point) for 68Ga-[APSC]2 radiotracer and 24 h p.i. for 67Ga-[APSC]2 are shown in Table 1. The biodistribution data indicate that the radiolabeled compound showed high initial uptake in the blood that reduced with time. At 4 h p.i., only 3.45% ID/g was presented in the blood circulation. The uptake in the major organs such as the liver, lungs, heart, spleen, and kidneys was also high at 1 and 2 h p.i. but radioactivity was cleared by these organs and no significant retention of radioactivity was observed at 24 h p.i., with the exception of kidneys which retained somewhat high radioactivity (up to 6.15% ID/g) at 24 h p.i. The uptake in the tumor was somewhat moderate at 1 h p.i but reached the maximum (% ID/g) at 2 h p.i. At 4 and 24 h p.i., 4.08% ID/g and 2.22% ID/g of radioactivity, respectively, were found in the tumor indicating a good retention of the radioactivity by the MDA-MB-231 tumors. The tumor-to-muscle ratio obtained at 2 h p.i. was 7.52. It is worth mentioning here that the tumor uptake value obtained with this radiotracer is found to be similar to the uptake profile reported earlier for 67Ga-thiosemicarbazone  and the tumor uptake washed out after 24 h p.i. indicating the opposite to the free 68Ga(III) cation that usually increased with time. However, the accumulation of 68Ga-[APSC]2 tracer in most of the other organs revealed that it is at least twofold lower than the tracer 67Ga-thiosemicarbazone . Thus, the good tumor targeting capabilities together with the favorable biodistribution profile of 68Ga-[APSC]2 warrant further evaluation and may tempt one to infer that this PET radiotracer may be useful as a molecular probe for detecting and staging of hypoxic tumors and their metastasis as well as monitoring tumor response to treatment.
|The values are average of % injected dose/gram ± SD for .|
Animals were injected with 67Ga-[APSC]2 radiotracer.
68Ga-[APSC]2 was synthesized in quantitative radiochemical yield and purity in less than 60 min. Biodistribution results of 68Ga-[APSC]2 in nude mice model bearing human MDA-MB-231 cell line xenografts demonstrated significant tumor uptake at 2 h p.i. and favorable pharmacokinetics over previously investigated 67Ga-[APSC]2 radiotracer. These results demonstrate that 68Ga-[APSC]2 radiotracer may be useful as PET probe for detecting and staging of hypoxic tumor; however, further evaluation is warranted.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
The research team would like to acknowledge the King Faisal Specialist Hospital and Research Center (KFSH&RC) and the International Atomic Energy Agency (IAEA) for their support. This research project was partially supported by a grant from the “Research Center of the Center for Female Scientific and Medical Colleges,” Deanship of Scientific Research, King Saud University. Also, special thanks are due to the comparative medicine department for their support in animal studies.
- V. B. Arion, M. A. Jakupec, M. Galanski, P. Unfried, and B. K. Keppler, “Synthesis, structure, spectroscopic and in vitro antitumour studies of a novel gallium(III) complex with 2-acetylpyridine 4N-dimethylthiosemicarbazone,” Journal of Inorganic Biochemistry, vol. 91, no. 1, pp. 298–305, 2002.
- I. C. Mendes, M. A. Soares, R. G. dos Santos, C. Pinheiro, and H. Beraldo, “Gallium(III) complexes of 2-pyridineformamide thiosemicarbazones: cytotoxic activity against malignant glioblastoma,” European Journal of Medicinal Chemistry, vol. 44, no. 5, pp. 1870–1877, 2009.
- T. S. Lobana, R. Sharma, G. Bawa, and S. Khanna, “Bonding and structure trends of thiosemicarbazone derivatives of metals-an overview,” Coordination Chemistry Reviews, vol. 253, no. 7-8, pp. 977–1055, 2009.
- J. Chan, A. L. Thompson, M. W. Jones, and J. M. Peach, “Synthesis and structural studies of gallium(III) and indium(III) complexes of 2-acetylpyridine thiosemicarbazones,” Inorganica Chimica Acta, vol. 363, no. 6, pp. 1140–1149, 2010.
- E. Bermejo, R. Carballo, A. Castineiras et al., “Synthesis, structural characteristics and biological activities of complexes of ZnII, CdII, HgII, PdII, and PtII with 2-acetylpyridine 4-methylthiosemicarbazone,” European Journal of Inorganic Chemistry, vol. 6, pp. 965–973, 1999.
- B. K. Rai, P. Choudhary, S. Rana, and P. Sahi, “Structural characterization and antifungal studies of some mixed ligand schiff base complexes with 6-bromo-2- thio 3-phenyl quinazoline-4(3H) one thiosemicarbazone,” Oriental Journal of Chemistry, vol. 23, pp. 271–275, 2007.
- M. C. Rodríguez-Argüelles, P. Tourón-Touceda, R. Cao et al., “Complexes of 2-acetyl-γ-butyrolactone and 2-furancarbaldehyde thiosemicarbazones: antibacterial and antifungal activity,” Journal of Inorganic Biochemistry, vol. 103, no. 1, pp. 35–42, 2009.
- D. X. West, A. M. Stark, G. A. Bain, and A. E. Liberia, “Copper(II) complexes of 2-formyl-, 6-methyl-2-formyl- And 2-benzoylpyridine N(4)-(2-methylpyridinyl)-,N(4)-(2-ethylpyridinyl)- and N(4)-methyl(2-ethylpyridinyl)thiosemicarbazones,” Transition Metal Chemistry, vol. 21, no. 4, pp. 289–295, 1996.
- B. S. Yadav, V. Kumar, and M. K. Yadav, “Vibrational spectra, thermodynamic parameters and fungicidal activity of 2-hydroxy-3-methoxybenzaldehyde thiosemicarbazone,” Indian Journal of Pure and Applied Physics, vol. 36, no. 10, pp. 557–566, 1998.
- J. Easmon, G. Pürstinger, G. Heinisch et al., “Synthesis, cytotoxicity, and antitumor activity of copper(II) and iron(II) complexes of 4N-azabicyclo[3.2.2]nonane Thiosemicarbazones derived from acyl diazines,” Journal of Medicinal Chemistry, vol. 44, no. 13, pp. 2164–2171, 2001.
- R. A. Finch, M.-C. Liu, A. H. Cory, J. G. Cory, and A. C. Sartorelli, “Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone; 3-AP): an inhibitor of ribonucleotide reductase with antineoplastic activity,” Advances in Enzyme Regulation, vol. 39, pp. 3–12, 1999.
- N. Adonai, K. N. Nguyen, J. Walsh et al., “Ex vivo cell labeling with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 5, pp. 3030–3035, 2002.
- Y. Fujibayashi, H. Taniuchi, Y. Yonekura, H. Ohtani, J. Konishi, and A. Yokoyama, “Copper-62-ATSM: a new hypoxia imaging agent with high membrane permeability and low redox potential,” Journal of Nuclear Medicine, vol. 38, no. 7, pp. 1155–1160, 1997.
- Y. Fujibayashi, C. S. Cutler, C. J. Anderson et al., “Comparative studies of Cu-64-ATSM and C-11-acetate in an acute myocardial infarction model: ex vivo imaging of hypoxia in rats,” Nuclear Medicine and Biology, vol. 26, no. 1, pp. 117–121, 1999.
- F. Dehdashti, M. A. Mintun, J. S. Lewis et al., “In vivo assessment of tumor hypoxia in lung cancer with 60Cu-ATSM,” European Journal of Nuclear Medicine and Molecular Imaging, vol. 30, no. 6, pp. 844–850, 2003.
- F. Dehdashti, P. W. Grigsby, J. S. Lewis, R. Laforest, B. A. Siegel, and M. J. Welch, “Assessing tumor hypoxia in cervical cancer by PET with 60Cu- labeled diacetyl-bis(N4-methylthiosemicarbazone),” Journal of Nuclear Medicine, vol. 49, no. 2, pp. 201–205, 2008.
- J. S. Lewis, T. L. Sharp, R. Laforest, Y. Fujibayashi, and M. J. Welch, “Tumor uptake of copper-diacetyl-bis (n4-methylthiosemicarbazone): effect of changes in tissue oxygenation,” Journal of Nuclear Medicine, vol. 42, no. 4, pp. 655–661, 2001.
- F. Haghighi Moghadam, A. R. Jalilian, A. Nemati, and M. Abedini, “Preparation and biodistribution studies of [67Ga]2- acetylpyridine 4,4-dimethyl thiosemicarbazone complex as a possible SPECT tracer for detection of malignancies,” Journal of Radioanalytical and Nuclear Chemistry, vol. 272, no. 1, pp. 115–121, 2007.
- A. R. Jalilian, H. Yousefnia, K. Shafaii, A. Novinrouz, and A. A. Rajamand, “Preparation and biodistribution studies of a radiogallium-acetylacetonate bis (thiosemicarbazone) complex in tumor-bearing rodents,” Iranian Journal of Pharmaceutical Research, vol. 11, no. 2, pp. 523–531, 2012.
- A. R. Jalilian, P. Mehdipour, M. Akhlaghi, H. Yousefnia, and K. Shafaii, “Evaluation of a [67Ga]-thiosemicarbazone complex as tumor imaging agent,” Scientia Pharmaceutica, vol. 77, no. 2, pp. 343–354, 2009.
- S. Delagrange, R. Delgado, and F. Nepveu, “Mn2+, Co2+, Cu2+ and Zn2+ complexes with two macrocyclic ligands bearing L-lactate-like functions: potentiometric studies and evaluation of superoxide-scavenging properties of the Mn2+ complex,” Journal of Inorganic Biochemistry, vol. 81, no. 1-2, pp. 65–71, 2000.
- P.-D. Pilar, P. Souza, J. R. Masaguer, and A. Arquero, “Complexes of 2-acetylpyridinesemicarbazone and 2-acetylpyridinethiosemicarbazone with cobalt(II), chromium(III) and copper(II),” Transition Metal Chemistry, vol. 12, no. 3, pp. 200–202, 1987.
- W. A. P. Breeman, M. De Jong, E. De Blois, B. F. Bernard, M. Konijnenberg, and E. P. Krenning, “Radiolabelling DOTA-peptides with 68Ga,” European Journal of Nuclear Medicine and Molecular Imaging, vol. 32, no. 4, pp. 478–485, 2005.
- K. P. Zhernosekov, D. V. Filosofov, R. P. Baum et al., “Processing of generator-produced 68Ga for medical application,” Journal of Nuclear Medicine, vol. 48, no. 10, pp. 1741–1748, 2007.
- Guide for the Care of and Use of Laboratory Animals, National Academy Press, Washington, DC, USA, 1996.
- L. Li and Y. Lu, “Inhibition of hypoxia-induced cell motility by p16 in MDA-MB-231 breast cancer cells,” Journal of Cancer, vol. 1, no. 1, pp. 126–135, 2010.
- C. L. Morton and P. J. Houghton, “Establishment of human tumor xenografts in immunodeficient mice,” Nature Protocols, vol. 2, no. 2, pp. 247–250, 2007.
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