Bimodal Quantitative Monitoring for Enzymatic Activity with

Jul 11, 2011 - Bimodal Quantitative Monitoring for Enzymatic Activity with Simultaneous Signal Increases in 19F ... The enzymatic activity and reactio...
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Bimodal Quantitative Monitoring for Enzymatic Activity with Simultaneous Signal Increases in 19F NMR and Fluorescence Using Silica Nanoparticle-Based Molecular Probes Kazuo Tanaka, Narufumi Kitamura, and Yoshiki Chujo* Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan ABSTRACT: We describe the bimodal quantitative assay for enzymatic activity in 19F NMR spectroscopy and fluorescence spectroscopy using a nanoparticle-based molecular probe. Perfluorinated dendrimers were tethered on silica nanoparticles with a phosphate-caged fluorescein as a linker. Before enzymatic reaction, the molecular rotation of the perfluorinated dendrimers should be highly restricted, and the 19F NMR signals from the perfluorinated dendrimers were too broad to be detected relative to the noise level. Fluorescence signals of fluorescein were suppressed by the presence of the diphosphate groups. Following the enzymatic reaction with an alkaline phosphatase, perfluorinated dendrimers and fluorescein were released, and the NMR signals of perfluorinated dendrimers and strong fluorescence from fluorescein were correspondingly observed. The enzymatic activity and reaction rates of the hydrolysis of alkaline phosphatase were detected from the increases of fluorescence and 19F NMR signals. Finally, the feasibility of the probe in the presence of miscellaneous molecules under biomimetic conditions was demonstrated by determining of the enzymatic activity in cell lysate. Quantitative analysis using both 19F NMR spectroscopy and fluorescence spectroscopy can be accomplished.

’ INTRODUCTION Magnetic resonance imaging (MRI) contrast agents enable us to receive information from the spots deep inside vital bodies with site and time specificities. Next, interest has been directed to developing the advanced imaging protocols or contrast agents which realize the quantitative assay as executed in chemical exchange saturation transfer MRI.1 3 A variety of fluorinated compounds have recently been used as a versatile platform to construct functional 19F MR probes4 7 for monitoring biological events such as gene expression,8 protein distribution,9,10 cell activity11 13 or enzymatic reaction,14 environmental alteration,15 18 and biological reactions.19 In addition, bimodal imaging probes which show corresponding19,20 or complementary signal changes21,22 between 19F NMR spectroscopy and another modality have been developed. However, very few examples have been reported to apply 19F MRI for quantitative assays. A “turn-on” probe that can show the enhanced signal after the target detection throughout all conditions may have better measurement precision. The kinetic information can be readily calculated from signal increases per unit time. The noises caused by the intrinsic signals from unreacted probes or background can be excluded by adjusting the threshold level. These advantages may enhance the simplicity of the measurements. Moreover, bimodal turn-on probes which offer simultaneous increases of the magnitudes of the respective signals between different modalities during the detections have potential advantages such as data reliability and yet also have potential limitations such as protein aggregation that affects both signaling systems, so that r 2011 American Chemical Society

bimodal probes deserve to be investigated in order to maximize advantages and minimize disadvantages. Herein, we report bimodal probes with 19F NMR spectroscopy and fluorescence spectroscopy for the quantitative assay of an enzymatic reaction. Perfluorinated dendrimers were anchored on silica nanoparticles (NPs) with the phosphate-caged fluorescein (Scheme 1). On silica NPs, the molecular rotation of the perfluorinated dendrimers should be highly restricted, and the NMR signals from the perfluorinated dendrimers can be suppressed.23 During enzymatic reaction with an alkaline phosphatase (AP), the NMR signals from perfluorinated dendrimers would be enhanced due to recovery of the molecular rotation, increasing the fluorescence emission from the released fluorescein. Consequently, the enzymatic activity and reaction rates of the hydrolysis of AP can be evaluated from the increases of fluorescence and 19F NMR signals. This is the first effort, to the best of our knowledge, to quantitatively evaluate a single enzymatic reaction with both 19F NMR and fluorescence spectroscopies.

’ EXPERIMENTAL SECTION General. 1H NMR and 13C NMR spectra were measured with

a JEOL EX-400 spectrometer operating at 400 MHz for 1H and 100 MHz for 13C. 19F and 29Si NMR spectra were measured with Received: August 27, 2010 Revised: June 23, 2011 Published: July 11, 2011 1484

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Scheme 1. Detection Mechanism for Enzymatic Reaction Using the NP Probes

a JEOL JNM-A400 spectrometer operating at 370 MHz for 19F and 80 MHz for 29Si. Coupling constants (J value) are reported in Hertz. The chemical shifts in 19F NMR are expressed in ppm downfield from trifluoroacetic acid as an external reference. Masses were determined with a MALDI-TOF mass spectroscopy (acceleration voltage 21 kV, negative mode) with 2,5-dihydroxybenzoic acid (DHB) as a matrix. Transmission electron microscopy (TEM) was performed using a JEOL JEM-100SX microscope operating at 100 kV. Emission spectra of the samples were monitored by a Perkin-Elmer LS50B fluorometer at 25 °C using a 1 cm path length cell. The excitation bandwidth was 0.1 nm. The emission bandwidth was 0.1 nm. Calf intenstine alkaline phosphatase (AP) was purchased from Invitrogen Corporation (USA). The dynamic light scattering (DLS) measurements were carried out at 90° scattering angle and 25 ( 0.2 °C using a FPAR1000 particle analyzer with a He Ne laser as a light source. The CONTIN program was used for data analysis to extract information on the average hydrodynamic radius (rH). F-POSS 2.19,22,23. To a suspension of octaammonium POSS24 26 (1, 1 g, 0.852 mmol) and ethyl trifluoroacetate (406 μL, 3.41 mmol) in methanol (20 mL), triethylamine (2 mL, 14.4 mmol) was added, and the reaction mixture was stirred at room temperature for 3 h. The resulting mixture was evaporated, and the crude was directly used in the next step. A clear oil was obtained after dialysis (895 mg, 83%); 1H NMR (D2O, 400 MHz) δ 3.26 (t, 8H, J = 7.0 Hz), 2.94 (t, 8H, J = 7.0 Hz), 1.70 (brs, 8H), 1.61 (brs, 8H), 0.64 (m, 16H); 13C NMR (D2O, 100 MHz) δ 155.9, 117.4, 41.52, 40.92, 21.76, 20.05, 8.77, 8.72; 29Si NMR (D2O, 80 MHz) δ 68.7, 68.4; 19F NMR (D2O, 373 MHz) δ 75.4; MALDI-TOF [(M+H)+], [POSS-TFA2] calcd. 1074.52, found 1073.29, [POSS-TFA3] calcd. 1170.53, found 1170.00, [POSS-TFA4]

calcd. 1266.53, found 1266.16, [POSS-TFA5] calcd. 1362.54, found 1361.76. Preparation of the Amino-Presenting Silica Nanoparticles.23,27 A solution containing 2 mL of tetraethoxysilane, 1 mL of 3-aminopropyltriethoxysilane, 7 mL of water, and 2 mL of ammonium hydroxide in 50 mL of ethanol was stirred at ambient temperature for 16 h, and then the white precipitate was separated by centrifugation. After washing with ethanol three times, the particles (117 ( 15 nm diameter from TEM, 280 ( 44 nm diameter from DLS) were obtained as a white powder. In this step, 960 nmol of trifluoroacetyl groups were attached to the particles. Preparation of the NP Probes. A solution containing 1.24 g of fluorescein (3.75 mmol) and POCl3 (20 mL) was refluxed for 2 h. The reaction was monitored by 1H NMR in CDCl3, and after finishing the reaction, the excess POCl3 was removed by evaporation. The brown residue containing 3 was used without purification for the next procedures. Identification was performed using the hydrolysis product of the compound 3 with 3,6fluorescein diphosphate in 1H NMR and MS measurements. F-POSS obtained above (0.852 mmol) and 3 were mixed in 20 mL of chloroform containing 2 mL of triethylamine (14.4 mmol). The phosphorylation of amino groups of F-POSS was monitored by the observation of the chemical shift at the 2position of the hydrogen atoms which exhibited a downfield shift after the amide formation. After finishing the reaction, 100 mg of the amino-presenting silica nanoparticles was added to the mixture. The reaction was monitored by ninhydrin reagents for consumption of the amino groups on the NPs. After stirring 1 h at room temperature, nanoparticles were centrifuged and washed with chloroform, water, and methanol. The white powder was obtained after drying (119 ( 16 nm diameter from TEM, 288 ( 46 nm 1485

dx.doi.org/10.1021/bc100381x |Bioconjugate Chem. 2011, 22, 1484–1490

Bioconjugate Chemistry diameter from DLS). To estimate the amount of F-POSS on the NP (81 nmol/mg), the 19F NMR spectrum of the solution containing the NP probe was dissolved in 1 N aqueous sodium hydroxide and compared to spectra of the standard samples with various concentrations of trifluoroacetic acid. The amount of fluorescein (471 nmol/mg) was estimated from the fluorescence intensity and UV absorption of the dissolved solutions. At the surface of the particles, 49% of the amino groups were tethered with fluorescein, and 34% with F-POSS. Enzymatic Reaction with the NP Probe. The NP probe (2.5 mg) was incubated in 500 μL of the reaction solution containing AP in 50 mM sodium phosphate, 100 mM Tris HCl, and 0.05 mM EDTA (pH = 7.0) at 37 °C. The reaction was terminated by centrifuging the reaction mixture, and then the supernatants were analyzed by 19F NMR. UV vis absorption measurements were performed with the diluted supernatants (10). Fluorescence measurements were executed with the diluted supernatants (1000). 19 F NMR Measurements for Determining Reaction Yields in Enzymatic Reactions and Relaxation Times. Relaxation times of F-POSS and trifluoroacetic acid in 19F NMR were taken with the following parameter sets: relaxation delay, 15 s; pulse width (90°), 51 μs; acquisition time, 88 ms; scan time, 4 times. 19 F longitudinal (T1) relaxation data were collected with ten delay times (1, 5, 10, 20, 50, 100, 250, 500, 1000, and 3000 ms) using a standard 1D inverse recovery pulse sequence. 19F transverse (T2) relaxation data were collected with 9 delay times (400, 500, 600, 700, 800, 900, 1000, 2000, and 8000 ms) using a standard 1D Carr-Purcel-Meiboom-Gill pulse sequence. Resonance intensities in relaxation experiments were measured and fit to an exponential function. The reaction yields were determined according to our previous work.23 19F NMR spectra for the analysis of synthetic compounds were taken at 25 °C with the following parameter sets: relaxation delay, 6 s; pulse width (45°), 12 μs; acquisition time, 88 ms; scan time, 8 times. In total, 1 min was required for 1D NMR acquisition. Standard solutions containing F-POSS with the concentration region 100 μM to 4 mM were prepared, and the linear relationship was determined between the concentration and the signal height of trifluoromethyl groups in F-POSS in the 19F NMR spectra. The reaction yields were determined by fitting the signal height to the standard line. Preparation for HeLa Cell Lysate. HeLa cells (1.0  106 cells) were cultured in a dish (90% confluent in ϕ100 mm dishes) and washed twice with ice-cold PBS( ). The cell lysate was then harvested with 2 mL of ice-cold CelLytic M Cell Lysis Reagent (Sigma-Aldrich, Inc., St. Louis, MO), kept at ambient temperature for 15 min, and centrifuged at 12 000 rpm for 5 min to remove the cell debris. The enzymatic volume activity (U/mL) of the supernatant was measured from the hydrolysis rate of p-nitrophenyl phosphate. The solutions (100 μL) containing 1 μL of the cell lysate, 6.7 mM p-nitrophenyl phosphate, and 2.0 mM magnesium chloride in 50 mM sodium phosphate buffer (pH = 6.0) were incubated at 37 °C, and the absorption changes at 405 nm were monitored. The volume activity was estimated from the fitting of the standard curve with CIAP.

’ RESULTS AND DISCUSSION Scheme 1 illustrates the bimodal detection mechanism using the NP-based probe for enzymatic activity in this study. The probe consists of three significant components, the signal unit,

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Scheme 2. Synthetic Sheme of the NP Pobesa

a

Reagents: (a) ethyl trifluoroacetate, triethylamine, methanol, 83%; (b) fluorescein, phosphorus oxytrichloride, triethylamine, chloroform, quant.; (c) F-POSS, chloroform; (d) amino-modified silica nanoparticles, chloroform.

the NP moiety, and the linker. As a 19F NMR signal unit, we used water-soluble perfluorinated polyhedral oligomeric silsesquioxane (F-POSS) which can be a good scaffold to construct functional 19F MR probes by modification with the signal regulation modulus because of high water-solubility and stability under biological conditions.19,22,23 The silica NPs work as a quencher for 19F NMR signals of the surface-tethered F-POSS molecules.23 On the surface of the NP, the molecular rotation of F-POSS should be restricted like in the solid phase, which suppresses the NMR signals. Indeed, the T1 and T2 relaxation times of fluorine atoms were determined to be 2.90 and 1.94 s in trifluoroacetic acid and 1.067 and 0.992 s in F-POSS, respectively. On the other hand, those values in the NP probes were not determined because of shorter transverse relaxation times and the low signal intensity of the 19F resonance. These results clearly indicate that the silica particles can accelerate the relaxation for 19 F NMR signals and work as a quencher for NMR signals. By the enzymatic digestion of the linker, F-POSS can be released, and the 19F NMR signals of F-POSS would be enhanced. To evaluate the AP activity, the diphosphate-caged fluorescein was used as the biodegradable linker between F-POSS and the surface of NPs. The 3,6-diphospholyrated fluorescein shows less fluorescence emission, and the intrinsic strong emission can be recovered after 1486

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Figure 2. (a) 19F NMR spectra of the supernatants after the incubation. The reactions were executed with the NP probes (F-POSS, 0.41 mM; F atom, 4.86 mM; fluorescein, 2.36 mM) in the presence or absence of AP (150 U/mL) in 50 mM sodium phosphate, 100 mM Tris HCl, and 0.05 mM EDTA (pH = 7.0) at 37 °C. (b) The fluorescence image of the 10-fold diluted supernatants before (left) and after 24 h incubation in the presence (center) or absence (right) of AP. The samples were irradiated at 365 nm with the transilluminator.

Figure 1. TEM images of (a) the amino-presenting silica NPs and (b) the F-POSS-coated silica NPs. The scale bar represents 500 nm length.

the elimination of both phosphate groups by AP.28 36 Hence, the enzymatic reaction can be assessed by the increases of two signal molecules, F-POSS and fluorescein, in 19F NMR and fluorescence measurements, respectively. F-POSS were synthesized using octaamino-POSS as a starting material by introducing trifluoroacetyl groups according to a previous report.23 We prepared amino-presenting silica NPs averaging 150 nm in diameter with the St€ober method.27 The amino groups of F-POSS were modified with the precursor of the diphosphate-caged fluorescein 3 to yield the compound 4 (Scheme 2). Because compounds 3 and 4 can be readily degraded, we used these compounds without purification for the next steps. Subsequently, the phosphoryl chloride groups of compound 4 were reacted with the amino groups on the surface of NPs. After washing with water and methanol thoroughly, the monodispersed modified NPs were obtained (Figure 1). From the measurements of the particle radii with TEM and DLS, the slight increases were observed. These data indicate that the interparticle cross-linking should hardly occur in the product. The amounts of F-POSS and fluorescein were determined by comparing the signal intensities with the standard samples as 81 nmol and 471 nmol on 1 mg of the NPs, respectively. The smaller existing ratio of F-POSS (1:5.8) than the theoretical value calculated from the reaction condition (1:4) indicates desorption of F-POSS from the particle surface during purification. This fact suggests that the phosphate-caged fluorescein not tethered to F-POSS should exist on the surface. The significant degradation

following undesired signal output or aggregation was not observed by adding the agent to BSA (1 mg/mL) or serum, or by altering the pH between pH 5 and 9. In addition, the signals were not observed after 24 h incubation at 37 °C at pH 7.0 in the presence of proteinase K. These results suggest that our probe could provide clear signals without the loss of sensitivity caused by unexpected interactions in vivo. A class of AP generally exists in the cell cytoplasm or surface in the whole body, and they can digest phosphodiester analogues without specificity.37,38 The AP activity is particularly enhanced in tumor regions.39,40 In addition, several tens to hundred nanometersized macromolecules can be specifically delivered to the tumor region by the enhanced permeability and retention (EPR) effect.41 47 Therefore, we can set the scenarios so that the APresponsive probes based on the nanoparticles can be expected to show enhanced signals after reaching tumor regions via the EPR effect or much vigorous AP activity. Hence, this system of assessing the AP activity could be a valid technique for early cancer diagnosis. 19F MRI is an insensitive detection method, so accumulation of the probe with sufficient concentrations would be necessitated for in vivo usages. Nanoparticle-based probes are advantageous because the EPR effect could possibly solve the problems on the sensitivity and the delivery of the probe. The reactivity of the probe toward AP was investigated. The reaction mixtures (0.5 mL) containing 5 mg/mL of the modified NPs (F-POSS, 0.41 mM; F atom, 4.86 mM; fluorescein, 2.36 mM) and AP (150 U/mL) in 50 mM sodium phosphate and 100 mM Tris HCl buffer (pH = 7.0) were incubated at 37 °C. 19F NMR signals and fluorescence intensity at 512 nm from the mixture were monitored. Both signals increased after incubation in the presence of AP (Figure 2). From the fitting to the standard line, the reaction yields were calculated as approximately 10% after 1 h incubation. On the other hand, signal intensities were continuously under the detection limit (less than 5% of the reaction 1487

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Figure 3. (a) Time-course of the intensity changes of 19F NMR signals from the supernatants after enzymatic reactions. The NP probes (2.5 mg) were incubated in 500 μL of the reaction solutions (F-POSS, 0.41 mM; F atom, 4.86 mM; fluorescein, 2.36 mM) containing various concentration of AP in 50 mM sodium phosphate, 100 mM Tris HCl, and 0.05 mM EDTA (pH = 7.0) at 37 °C. The reaction yields were monitored with 19F NMR and calculated by fitting on the standards. The errors represent the standard deviation calculated from the three data sets. (b) Time-courses of the emission changes at 512 nm from the 10fold diluted supernatants after reaction with various concentrations of AP. The errors represent the standard deviation calculated from the three data sets.

yield) after 24 h incubation in the absence of AP. These results indicate that the AP activity can be detected using our probe as the corresponding increases of 19F NMR and fluorescence signals as we designed. We executed the reactions with various concentrations of AP, and the signal intensities from the reaction mixtures were monitored with 19F NMR and fluorescence measurements (Figure 3). The increases of the signal intensities were compared with the standard line prepared with the concentration-definitive F-POSS solutions and converted as the reaction yields. The increases of the yields calculated from 19F NMR and fluorescence signals showed good agreement. In the presence of 200 U/mL of AP, the reactions reached a plateau after the 8 h incubation. The detection limits were correspondingly 50 U/mL of AP. These data suggest that enzymatic reaction can be assessed by the synchronized increases of fluorescence and 19F NMR signals. This fact proposes that the signal change of the probe in one modality could be corrected by the signal response in the other modality. The rapid increase of fluorescence intensity within 2 h could be caused from the fluorescein molecules tethered to the surface of the NPs directly and the amino-terminal of F-POSS. To demonstrate feasibility of the quantitative analysis of the probe in the presence of the miscellaneous molecules under

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Figure 4. Determination of AP activity in the HeLa cell lysate. (a) Fitting of the obtained value (clear triangular dot) from the 19F NMR measurements to the standard line according to the results of Figure 3a. (b) Fitting of the obtained value (clear triangular dot) from the fluorescence measurements to the standard line according to the results of Figure 3b. The plots represent the averages calculated from the three data sets.

biomimetic conditions, the reactions were performed in the HeLa cell lysate. Initially, the AP activity was calculated from the hydrolysis rate of p-nitrophenyl phosphate in the lysate, and it was determined to be 95 ( 9 U/mL. The linear increases of the magnitude of both the 19F NMR and fluorescence signal intensity were observed from our probes. Consequently, the enzymatic activities of the cell lysate were evaluated to be 97 (19F NMR) and 98 (fluorescence) U/mL from the slope fitted to the standard line, respectively (Figure 4). These data suggest that the NP probe can be used for bimodal quantitative assay with biological samples.

’ CONCLUSION In summary, we present here the bimodal detection in 19F NMR and fluorescence for a single enzymatic reaction. The NP probes can gradually release perfluorinated dendrimers and fluorescein with the progress of enzymatic reactions. The enzymatic activity can be evaluated from the increasing rates of the signal magnitudes in 19F NMR and fluorescence during the reaction. Finally, it was demonstrated that the enzymatic activity of the cell lysate evaluated with our probes showed good agreement with that with the conventional method. We conclude that this system could provide a feasible strategy for receiving quantitative information in MRI. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Fax: +81-75-3832605. Phone: +81-75-383-2604. 1488

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’ ACKNOWLEDGMENT This research was partly supported by Nakatani Foundation of Electronic Measuring Technology Advancement (for K.T.) and Grant-in-Aid for Young Scientists (B), no. 22750107, from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. ’ REFERENCES (1) Terreno, E., Castelli, D. D., Viale, A., and Aime, S. (2010) Challenges for molecular magnetic resonance imaging. Chem. Rev. 110, 3019–42. (2) Ali, M. M., Liu, G., Shah, T., Flask, C. A., and Pagel, M. D. (2009) Using two chemical exchange saturation transfer magnetic resonance imaging contrast agents for molecular imaging studies. Acc. Chem. Res. 42, 915–24. (3) Shapiro, M. G., Atanasijevic, T., Faas, H., Westmeyer, G. G., and Jasanoff, A. (2006) Dynamic imaging with MRI contrast agents: quantitative considerations. Magn. Reson. Imaging 24, 449–62. (4) Srinivas, M., Heerschap, A., Ahrens, E. T., Figdor, C. G., and de Vries, I. J. M. (2010) 19F MRI for quantitative in vivo cell tracking. Trends Biotechnol. 28, 363–70. (5) Morawski, A. M., Winter, P. M., Yu, X., Fuhrhop, R. W., Scott, M. J., Hockett, F., Robertson, J. D., Gaffney, P. J., Lanza, G. M., and Wickline, S. A. (2004) Quantitative “magnetic resonance immunohistochemistry” with ligand-targeted 19F nanoparticles. Magn. Reson. Med. 52, 1255–62. (6) Yu, J., Kodibagkar, V. D., Cui, W., and Mason, R. P. (2005) 19F: A versatile reporter for non-invasive physiology and pharmacology using magnetic resonance. Curr. Med. Chem. 12, 819–48. (7) Senanayake, P. K., Kenwright, A. M., Parker, D., and van der Hoorn, S. K. (2007) Responsive fluorinated lanthanide probes for 19F magnetic resonance spectroscopy. Chem. Commun. 2923–5. (8) Cui, W., Otten, P., Li, Y., Koeneman, K. S., Yu, J., and Mason, R. P. (2004) Novel NMR approach to assessing gene transfection: 4-Fluoro-2-nitrophenyl-β-D-galactopyranoside as a prototype reporter molecule for β-galactosidase. Magn. Reson. Med. 51, 616–20. (9) Takaoka, Y., Sakamoto, T., Tsukiji, S., Narazaki, M., Matsuda, T., Tochio, H., Shirakawa, M., and Hamachi, I. (2009) Self-assembling nanoprobes that display off/on 19F nuclear magnetic resonance signals for protein detection and imaging. Nat. Chem. 1, 557–61. (10) Higuchi, M., Iwata, N., Matsuba, Y., Sato, K., Sasamoto, K., and Saido, T. C. (2005) 19F and 1H MRI detection of amyloid β plaques in vivo. Nat. Neurosci. 8, 527–33. (11) Zimmermann, U., N€oth, U., Gr€ohn, P., Jork, A., Ulrichs, K., Lutz, J., and Haase, A. (2000) Non-invasive evaluation of the location, the functional integrity and the oxygen supply of implants: 19F nuclear magnetic resonance imaging of perfluorocarbon-loaded Ba2+-alginate beads. Artif. Cells Blood Substit. Immobil. Biotechnol. 28, 129–46. (12) Ahrens, E. T., Flores, R., Xu, H. Y., and Morel, P. A. (2005) In vivo imaging platform for tracking immunotherapeutic cells. Nat. Biotechnol. 23, 983–87. (13) Maki, J., Masuda, C., Morikawa, S., Morita, M., Inubushi, T., Matsusue, Y., Taguchi, H., and Tooyama, I. (2007) The MR tracking of transplanted ATDC5 cells using fluorinated poly-L-lysine-CF3. Biomaterials 28, 434–40. (14) Mizukami, S., Takikawa, R., Sugihara, F., Hori, Y., Tochio, H., W€alchli, M., Shirakawa, M., and Kikuchi, K. (2008) Paramagnetic relaxation-based 19F MRI probe to detect protease activity. J. Am. Chem. Soc. 130, 794–5. (15) Okada, S., Mizukami, S., and Kikuchi, K. (2010) Application of a stimuli-responsive polymer to the development of novel MRI probes. ChemBioChem 11, 785–7. (16) Tanabe, K., Harada, H., Narazaki, M., Tanaka, K., Inafuku, K., Komatsu, H., Ito, T., Yamada, H., Chujo, Y., Matsuda, T., Hiraoka, M., and Nishimoto, S. (2009) Monitoring biological one-electron reduction

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Bioconjugate Chemistry

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dx.doi.org/10.1021/bc100381x |Bioconjugate Chem. 2011, 22, 1484–1490