Shin
Mizukami
ab,
Hisashi
Matsushita
a,
Rika
Takikawa
a,
Fuminori
Sugihara
c,
Masahiro
Shirakawa
d and
Kazuya
Kikuchi
*ab
aDivision of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: kkikuchi@mls.eng.osaka-u.ac.jp; Fax: (+81) 6-6879-7875
bImmunology Frontier Research Center (IFReC), Osaka University, Osaka, 565-0871, Japan
cInternational Graduate School of Arts and Sciences, Yokohama City University, Kanagawa, 230-0045, Japan
dGraduate School of Engineering, Kyoto University, Kyoto, 615-8510, Japan
First published on 7th April 2011
Imaging of gene expression by magnetic resonance imaging (MRI) yields direct information regarding living systems that cannot be obtained via other methods. In this study, we report the rational design and synthesis of a novel 19F MRI probe that detects β-galactosidase (β-gal) activity, enabling the imaging of gene expression in cells. The 19F MRI signal of the probe was quenched by the intramolecular paramagnetic resonance enhancement from a Gd3+ ion. A contrivance was made in the probe structure to recover the 19F MRI signal after hydrolysis by β-gal with a following self-immolative reaction. This 19F MRI signal change was observed in the physiological aqueous condition. The probe could also detect β-gal activity in fixed HEK293T cells. In conclusion, this new probe enables the 19F MRI detection of cellular gene expression. The probe design strategy is also expected to lead to the development of MRI probes for a wide variety of hydrolase activities.
Recently, several smart 1H MRI probes for visualizing gene expressionvia β-galactosidase activity have been reported.6 In principle, however, such 1H MRI signal enhancement needs to be discriminated from the background 1H MRI signals of water, fatty acids, and other biomolecules. To avoid this limitation, we have focused on the use of 19F MRI. 19F, as well as 1H, is one of the most highly sensitive nuclei for NMR spectroscopy and MRI,7 and almost no intrinsic 19F MRI signals are observed in animal bodies. Thus, 19F MRI probes that can visualize biological events have been increasingly reported.8 We have also developed off-on switching 19F MRI probes to detect protease activity9 on the basis of paramagnetic relaxation enhancement (PRE),10 a phenomenon in which the relaxation of nuclei is enhanced near paramagnetic molecules.
By expanding this probe principle, we here report a novel 19F MRI probe that detects cellular gene expression. β-galactosidase (β-gal) was chosen as the reporter protein for gene expression, because it has several advantages where reporter proteins are concerned.3,11 The advantages are as follows: (1) induction of β-gal synthesis occurs over a large dynamic range, (2) β-gal is tolerated and functional in many organisms including mammals, (3) various substrates of β-gal are available or easily synthesized, (4) many assay methods that use β-D-galactopyranoside-coupled aglycones are available, and (5) there is almost no intrinsic β-gal activity in mammalian cells. Therefore, β-gal is one of the most widely used reporter proteins for imaging of gene expression. Through the detection of β-gal activity, we tried 19F MRI detection of cellular gene expression.
Fig. 1 Structure of Gd-DFP-Gal and the principle for the 19F MRI detection of β-gal activity. |
Another designed function of Gd-DFP-Gal is the self-immolative property that can be induced by enzymatic cleavage. When Gd-DFP-Gal is hydrolyzed by β-gal, the probe is expected to be automatically converted to the corresponding quinone methide by the successive elimination of the substituent at the benzyl position.13 Thus, the T2 of the trifluoromethyl group extends after the β-galactoside bond is cleaved because of the cancellation of the intramolecular PRE. MRI signal intensity (i.e., the peak height of the NMR signal) is proportional to exp(−t/T2), where t is the echo time in the spin-echo method. Thus, the T2 extension leads to an increase in the MRI signal. On the basis of this principle, we expected that the originally quenched 19F MRI signal of Gd-DFP-Gal would emerge upon the enzyme reaction.
Gd-DFP-Gal was synthesized in five steps (Scheme S1, ESI†). Details of the synthetic procedure are described in the Supporting Information.† As we expected, the NMR peak of Gd-DFP-Gal was not observed, although that of the Gd-free probe DFP-Gal was a sharp single peak (Fig. S2, ESI†). Disappearance of the 19F NMR peak of Gd-DFP-Gal indicates that the T2 was markedly reduced because of the strong intramolecular PRE.
Fig. 2 Detection of β-gal activity by Gd-DFP-Gal. (a) Time-dependent 19F NMR spectral change of Gd-DFP-Gal (1 mM) under incubation with β-gal. Sodium trifluoroacetate was used as the internal standard (0 ppm). (b) Confirmation of the enzymatic cleavage by RP-HPLC (eluent: H2O–acetonitrile containing 0.1% TFA). (c) Time course of the density-weighted 19F MR phantom images of Gd-DFP-Gal (1 mM) at 37 °C after β-gal was added. |
The relaxation times T1 and T2 of the reaction sample became 0.306 s and 0.086 s, respectively, after the enzyme reaction. Both of them showed considerable extension compared to those of Gd-DFP-Gal, probably due to the cancellation of the intramolecular PRE from Gd3+. These values are still less than those of the Gd3+-free probe DFP-Gal: 1.293 s for T1 and 0.271 s for T2. When the relaxation times of Gd-DFP-Gal were measured at various probe concentrations after the enzymatic cleavage, both T1 and T2 extended as the concentration decreased (Fig. S3 and Table S1, ESI†). This concentration dependency of the relaxation times indicates that the intermolecular PRE is effective under the experimental condition even after the enzyme reaction is complete. To confirm the probe specificity, Gd-DFP-gal was incubated with other similar enzymes, α-galactosidase and β-glucuronidase. However, 19F NMR signals of Gd-DFP-gal were not recovered by incubation with such enzymes (Fig. S4, ESI†).
To demonstrate the possibility of further application, 19F MRI detection of β-gal activity was performed using Gd-DFP-Gal. 19F MRI phantom images were measured using an 11.7 T MRI instrument. Gd-DFP-Gal was mixed with Escherichia coli β-gal before being poured into a 1-mm-inner radius capillary. The density-weighted MR images were then captured by the fast spin-echo method. As expected from the 19F NMR results, Gd-DFP-Gal showed no 19F MRI signals in the absence of β-gal. After the probe was mixed with β-gal, however, the 19F MRI signals gradually increased in a time-dependent manner (Fig. 2c). Without addition of the enzyme, the MRI image did not show any signals for several hours (data not shown). These results demonstrate that this novel mechanism-based probe Gd-DFP-Gal enables the specific 19F MRI detection of β-gal activity.
Fig. 3 19F NMR and 19F MRI detection of gene expression in HEK293T cells. (a) Illustration of the experimental procedures for the 19F NMR and 19F MRI measurements. (b) 19F NMR spectra of the culture medium containing 1 mM Gd-DFP-Gal incubated with fixed cells expressing (top) or not expressing (bottom) β-gal. (c) 1H (left) and 19F (right) MR images of culture vessels containing 1 mM Gd-DFP-Gal fixed cells. Color scale bars were inserted in the images. |
Then, the 19F MRI detection of β-gal gene expression was attempted. HEK293T cells expressing or not expressing β-gal were cultured on 7-mm-diameter glass vessels. After the fixation of the cells, Gd-DFP-Gal (final conc.: 1 mM) was added into the glass vessels, and the cells were incubated at 37 °C for 2 h. The vessels were stacked in an 8 mm NMR tube, as shown in Fig. 3b, and the 1H and 19F MR images were captured. Although both vessels showed indistinguishable signal intensity in 1H MRI (Fig.3c left), only the vessel that included HEK293T cells expressing β-gal showed remarkable 19F MRI signals (Fig. 3c right). These results indicate that Gd-DFP-Gal can specifically detect gene expression in fixed HEK293T cells by means of reporter β-gal activity.
Fig. 4 Two 19F MRI probe design strategies using PRE cancellation by (a) enzymatic cleavage of the substrate linker, and (b) enzyme activity-induced self-immolative reaction. |
Although imaging of gene expression in fixed cells by using Gd-DFP-Gal, there are two obstacles for future perspective to in vivo imaging. One is the membrane permeability of the probes. Since the new probe did not permeate cell membrane, the cells needed to be fixed with formaldehyde for imaging. However, use of cell-penetrating peptides,14 which enabled the incorporation of proteins into live cells, may dissolve the problem.
The other is the sensitivity. Generally, the sensitivity of 19F MRI probes is worse than 1H MRI probes. This is because 1H MRI visualizes many water molecules around the probe molecules, although 19F MRI probes give only the probe signals. Concerning the problem, improvement of both probes and instruments will contribute to solve it. About the probe sensitization, we are under the development and would report elsewhere in future.
For 19F NMR analysis, the cells were cultured with 1 mM Gd-DFP-Gal for 2 h at 37 °C in the reaction buffer (10 mM Tris-sodium buffer (pH 7.3) and 10 mM magnesium chloride) on 24-well plates. Then, the supernatants were moved into NMR tubes and the 19F NMR spectra were measured.
For 19F MRI analysis, the cells were moved onto 7 mm (outer diameter) glass vessels (Hilgenberg GmbH), and were incubated for 7 h at 37 °C in DMEM with 10% FBS. After the cells were washed three times with PBS, they were incubated with 3.7% formaldehyde for 10 min at room temperature. Then, cells were washed three times with PBS and incubated with 1 mM Gd-DFP-Gal for 2 h at 37 °C in the reaction buffer (Tris-sodium buffer (pH 7.3) and 10 mM magnesium chloride). The vessels were put into an 8 mm NMR tube, and the 1H and 19F MRI were measured.
Footnote |
† Electronic supplementary information (ESI) available: Synthesis of compounds, representative HPLC chromatograms and 19F NMR. See DOI: 10.1039/c1sc00071c |
This journal is © The Royal Society of Chemistry 2011 |