Dual 19F/1H MR gene reporter molecules for in vivo detection of β-galactosidase

Article abstract of DOI:10.1021/bc200647q

Increased emphasis on personalized medicine and novel therapies requires the development of noninvasive strategies for assessing biochemistry in vivo. The detection of enzyme activity and gene expression in vivo is potentially important for the characterization of diseases and gene therapy. Magnetic resonance imaging (MRI) is a particularly promising tool, since it is noninvasive and has no associated radioactivity, yet penetrates deep tissue. We now demonstrate a novel class of dual ¹H/¹⁹F nuclear magnetic resonance (NMR) lacZ gene reporter molecule to specifically reveal enzyme activity in human tumor xenografts growing in mice. We report the design, synthesis, and characterization of six novel molecules and evaluation of the most effective reporter in mice in vivo. Substrates show a single ¹⁹F NMR signal and exposure to β-galactosidase induces a large ¹⁹F NMR chemical shift response. In the presence of ferric ions, the liberated aglycone generates intense proton MRI T₂ contrast. The dual modality approach allows both the detection of substrate and the imaging of product enhancing the confidence in enzyme detection.

Full text of DOI:10.1021/bc200647q

Article
pubs.acs.org/bc
19
Dual F/1H MR Gene Reporter Molecules for in Vivo Detection of
β-Galactosidase
Jian-Xin Yu, Vikram D. Kodibagkar,† Rami R. Hallac, Li Liu, and Ralph P. Mason*
Department of Radiology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
S
* Supporting Information
ABSTRACT: Increased emphasis on personalized medicine
and novel therapies requires the development of noninvasive
strategies for assessing biochemistry in vivo. The detection of
enzyme activity and gene expression in vivo is potentially
important for the characterization of diseases and gene
therapy. Magnetic resonance imaging (MRI) is a particularly
promising tool, since it is noninvasive and has no associated
radioactivity, yet penetrates deep tissue. We now demonstrate
a novel class of dual 1H/19F nuclear magnetic resonance
(NMR) lacZ gene reporter molecule to specifically reveal
enzyme activity in human tumor xenografts growing in mice.
We report the design, synthesis, and characterization of six
novel molecules and evaluation of the most effective reporter
in mice in vivo. Substrates show a single 19F NMR signal and exposure to β-galactosidase induces a large 19F NMR chemical shift
response. In the presence of ferric ions, the liberated aglycone generates intense proton MRI T2 contrast. The dual modality
approach allows both the detection of substrate and the imaging of product enhancing the confidence in enzyme detection.
spectroscopy would define substrate accumulation and
conversion, while iron based H MRI contrast reveals regional
enzyme activity.
INTRODUCTION
■
1
Several reporter proteins have been used in gene expression
and regulation studies including β-galactosidase (β-gal), firefly
luciferase (luc), β-glucuronidase, fluorescent proteins (such as
green fluorescent protein, GFP), transferrin and ferritin, the
enzymes creatine and arginine kinase, tyrosinase, and poly-
cations such as poly lysine.1,2 Historically, the lacZ gene en-
coding β-gal has been widely used with applications ranging
from molecular biology to small animal investigations and
clinical trials including assays of clonal insertion, transcriptional
activation, protein expression, and protein interaction.3,4 Re-
cently, various innovative approaches to assessing β-gal activity
in vivo have been presented exploiting gadolinium contrast en-
hanced 1H magnetic resonance imaging (MRI) or 19F
NMR,5−12 optical,13−18 and radionuclide imaging.19−21 Tradi-
tional 19F NMR spectroscopy approaches have the advantage of
detecting both the substrate and the product simultaneously,7−9
but while imaging is feasible,22,23 the achievable signal-to-noise
has so far been insufficient for imaging in animals. By com-
Escherichia coli (lacZ) β-gal catalyzes the hydrolysis of
β-D-galactopyranosides by cleavage of the C−O bond with
β-configuration between D-galactose and the aglycone. Considering
the multiple requirements for an enzyme responsive 19F/1H MRI
indicator, we chose salicylaldehyde nicotinoyl hydrazone, sali-
cylaldehyde isonicotinoyl hydrazone, and salicylaldehyde benzoyl
hydrazone as aroylhydrazone chelators based on reported
characteristics.26−28 A fluorine atom introduced at the ortho or
para position to the C1(gal)-O bond in the salicylaldehyde frag-
ment was expected to display a large 19F NMR chemical shift in
response to cleavage by β-gal.29
We now report proof of principle using a fluorosalicylalde-
hyde aroylhydrazone β-D-galactopyranoside, which provides a
19F NMR signal sensitive to β-gal enzyme cleavage and the
liberated aglycone spontaneously traps ferric ions generating 1H
MRI contrast on T2-weighted images (Figure 1). We currently
add ferric ammonium citrate, but contrast could, in principle,
develop from entrapment of endogenous Fe3+.
1
parison, H MRI contrast can be far more sensitive, but the
presence of substrate and the formation of product may not
be readily identifiable due to tissue heterogeneity. We have
demonstrated an Fe(III)-based H MRI approach using the
commercially available black histological stain sodium 3,4-
cyclohexenoesculetin-β-D-galactopyranoside (S-Gal sodium
salt) to detect β-gal activity in stably transfected lacZ expressing
cancer cells in vitro and in vivo and this approach was also used
to label and track stem cell localization.24,25 It occurred to us
that the approaches could be combined, whereby 19F NMR
EXPERIMENTAL PROCEDURES
Detailed molecular characterization results based on chemical
shift assignment and chemical analyses are provided in
1
■
Received: November 30, 2011
Revised: February 17, 2012
Published: February 18, 2012
© 2012 American Chemical Society
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1
Figure 1. Proposed mode of action: β-galactosidase activity is detected by 19F NMR chemical shift accompanying release of aglycone and H MRI
contrast induced by ferric ion complex.
Figure 2. Reactions and the structures of 1−17. Reaction conditions: (a) CH2Cl2−H2O, pH 8−9, 50 °C, TBAB, N2, 5−6 h, 92% (→4) or 86% (→
5), respectively; (b) EtOH, AcOH (20 μL), benzoic hydrazide, nicotinic hydrazide or isoniazide (1.1 equiv), 80 °C, N2, 4−5 h, 100% (→6), 90% (→
7), 95% (→8), 95% (→9), 100% (→10), and 93% (→11) respectively; (c) 0.5 M NH3-MeOH, 0 °C→r.t., 24 h, quantitative yields.
Supporting Information. All in vivo studies were performed
with approval from the Institutional Animal Care and Use
Committee.
with CH2Cl2 (4 × 25 mL), washed, dried (Na2SO4), evaporated
under reduced pressure to give a syrup, which was purified by
column chromatography on silica gel to give 2-[(2′,3′,4′,6′-tetra-O-
acetyl-β-D-galactopyranosyl)oxy]-3 or 5-fluorobenzaldehydes
4−5, as white crystals in >85% yield.
The synthetic route and structures of 1−17 are shown in
Figure 2. Reaction of 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl
bromide 1 (Sigma Chemical Company, St Louis, MO) with 3- or
5-fluorosalicylaldehydes 2 or 3 at 50 °C catalyzed by tetra-
butylammonium bromide (TBAB) in an aqueous dichloro-
methane biphasic system (pH 8−9) under N2 afforded
2-[(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)oxy]-3 or 5-fluoro-
benzaldehydes 4 or 5 in high yields (82−92%). Treatment of
4 or 5, respectively, with 1.1 equiv of benzoic hydrazide, nicotinic
hydrazide, or isoniazid in acidic medium at 80 °C produced the
corresponding 2-[(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)-
oxy]-3 or 5-fluorobenzaldehyde aroylhydrazones 6−11 in 90−
100% yields, which were deacetylated with NH3/MeOH from
0 °C to room temperature giving their free galactopyranosides
12−17 in quantitative yields.
Detailed Syntheses. 2-[(2′,3′,4′,6′-Tetra-O-acetyl-β-D-
galactopyranosyl)oxy]-3 or 5-Fluoro-benzaldehydes 4−5.
General Procedure. A solution of 2,3,4,6-tetra-O-acetyl-α-D-
galactopyranosyl bromide 1 (615 mg, 1.5 mmol) in CH2Cl2
(10 mL) was added dropwise to a vigorously stirred biphasic
mixture (pH 8−9) of 3- or 5-fluorosalicylaldehyde (2−3) (252 mg,
1.8 mmol) and tetrabutylammonium bromide (TBAB) (160 mg,
0.5 mmol) in CH2Cl2−H2O (20 mL, 1:1 v/v) over a period of 1 h
at 50 °C under N2, and the stirring continued for 4−5 h until
TLC showed complete reaction. The products were extracted
2-[(2′,3′,4′,6′-Tetra-O-acetyl-β-D-galactopyranosyl)oxy]-3
or 5-Fluorobenzaldehyde Aroylhydrazones 6−11. General
Procedure. A solution of 2-[(2′,3′,4′,6′-tetra-O-acetyl-β-D-gala-
ctopyranosyl)oxy]-3 or 5-fluorobenzaldehyde 4 or 5 (200 mg,
0.44 mmol) in anhydrous EtOH (15 mL) containing AcOH
(20 μL) was stirred vigorously respectively with benzoic hydra-
zide, nicotinic hydrazide, or isoniazid (0.48 mmol, 1.1 equiv) at
80 °C under N2 until TLC showed that the reaction was com-
plete, then coevaporated with toluene to dryness in vacuo. Crys-
tallization from EtOH-H2O or chromatography of the crude
syrup on silica gel with appropriate eluents yielded the corres-
ponding 2-[(2′,3′,4′,6′-tetra-O-acetyl-β-D-galactopyranosyl)oxy]-3
or 5-fluorobenzaldehyde aroylhydrazones 6−11 in 90−100%
yields.
2-[(β-D-Galactopyranosyl)oxy]-3 or 5-Fluorobenzaldehyde
Aroylhydrazones 12−17. General Procedure. A solution of
2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]-3 or 5-fluoro-
benzaldehyde aroylhydrazones 6−11 (200 mg) in anhydrous
MeOH (20 mL) containing 0.5 M NH3 was vigorously stirred
from 0 °C to room temperature overnight until TLC showed com-
plete reaction, then evaporated to dryness in vacuo. Chromatog-
raphy of the crude syrup on silica gel with EtOAc-MeOH afforded
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the corresponding free β-D-galactopyranosides 12−17 in quanti-
tative yields.
In Vivo MR Studies. PC3 cells (wild type or transfected to
stably express lacZ8) were implanted subcutaneously in thighs
of SCID mice (n = 3). NMR studies were performed at 4.7 T
using a Varian Unity INOVA horizontal bore scanner
3-Fluorosalicylaldehyde Nicotinoyl Hydrazone (3-FBNH)-
Hydrolysis Product of 13. A solution of 3-fluorosalicylaldehyde
(330 mg, 2.36 mmol) in anhydrous EtOH (15 mL) containing
AcOH (20 μL) was stirred vigorously with nicotinic hydrazide
(323 mg, 2.36 mmol) at 80 °C under N2 until TLC showed
that the reaction was complete, then coevaporated with toluene
to dryness in vacuo. Crystallization from EtOH-H2O yielded
3-FBNH (526 mg) as white needles.
Fe:3-FBNH Complex. To a solution of 3-FBNH (500 mg,
1.93 mmol) and Et3N (195 mg, 1.93 mmol) in anhydrous MeOH
(100 mL) was added dropwise a solution of Fe(ClO4)3·6H2O
(446 mg, 0.97 mmol) in anhydrous MeOH (50 mL) with stirring
and gentle reflux under N2 for 30 min. Upon cooling, a fine black
precipitate was obtained, which was filtered, washed with EtOH
then Et2O, and dried in vacuo yielding Fe-3-FBNH Complex
[Fe(3-FBNH-H)2](ClO4)·3H2O (576 mg) as black powder.
Anal. Calcd. for C26H24ClFeN6O11F2 (%): C, 43.03, H, 3.34, N,
11.59; Found: C, 42.95, H, 3.41, N, 11.48.
1
(200.1 MHz for H, 188.2 MHz 19F). When the tumors reach-
ed ∼0.8 cm in diameter, mice were anesthetized (isoflurane/
air) and placed on a platform with the tumor-bearing leg
inserted into a 2-cm-diameter home-built volume coil (tunable
1
from 200.1 MHz for H to 188.2 MHz for 19F). The animal
temperature was maintained at 37 °C by a warm pad with
circulating water. The mouse bed was inserted into the bore of
the MR scanner and shimming was performed on the tissue
water proton signal. T1 and T2 maps of the tumor were mea-
sured using a spin echo sequence with varying repetition and
echo times (acquisition parameters: field of view (FOV) 50 mm
× 50 mm, matrix 128 × 128, slice thickness 1 mm, 15 slices).
The mouse-bearing platform was removed from the magnet
and a solution of 13 (50 μL 50 mM, DMSO/PBS 1:1 v/v) was
injected directly into the tumor in a “fan” pattern. The platform
was replaced in the magnet and 19F NMR spectra were ob-
tained immediately after retuning the coil to the 19F resonance
frequency. Each spectrum was acquired in 166 s (acquisition
parameters: pulse width 45 μs, 128 acquisitions, spectral width
100 ppm, 60 Hz exponential line broadening). The mouse-
bearing platform was again removed from the magnet and a
solution of FAC (50 μL 50 mM, PBS) was now injected
directly into the tumor in a similar pattern to 13. 19F NMR
Detection of β-Galactosidase by 19F NMR in Solution. 2-
[(β-D-galactopyranosyl)oxy]-3-fluorobenzaldehyde nicotinoyl
hydrazone 13, 2-[(β-D-galactopyranosyl)oxy]-3-fluorobenzalde-
hyde isonicotinoyl hydrazone 14 (2.11 mg, 5.0 μmol), or
2-[(β-D-galactopyranosyl)oxy]-5-fluorobenzaldehyde benzoyl-
hydrazone 15 (2.10 mg, 5.0 μmol) were dissolved in PBS
(595 μL) and a solution of β-gal (5 μL, 1 unit/μL; E801A,
Aldrich) was added. 19F NMR time course spectra were ac-
quired immediately at 376 MHz in 102 s each and continued at
37 °C over 4 h to assess relative rates of activity. 13 was also
examined by 19F MRI. In this case, 13 (7 mg) was dissolved in
PBS/DMSO (250 μL 1:1 mixture) at 37 °C and β-galactosidase
(E801A 20 units) added. 19F MR images were acquired at
376 MHz with 930 μm in plane resolution across a 30 mm ×
30 mm field of view and 10 mm slice thickness in 4 min 16 s
each using 8 averages at each phase encode and TR = 1000 ms,
TE = 1.6 ms, and 90° flip angle.
1
spectra were obtained and the coil was retuned to the H
frequency, and new T1 and T2 maps were acquired. For analysis,
regions with injected contrast agent were identified based on
the T2- and T1-weighted images (to delineate tumor boundary
and locate the injected Fe3+ ions, respectively). For one animal,
no injection site could be identified inside the tumor and hence
the data for this tumor were not used. To confirm β-gal activity
in tumors, tissues were embedded in Tissue-Tek OCT (Miles
Laboratory, Elkhart, IN, USA), and frozen in liquid nitro-
gen. Cryostat sections (8 μm) were collected on gelatin-coated
glass slides and stained with X-gal and eosin (Sigma) for β-gal
activity.
Testing Product Aglycone Visibility in Vivo. A mixture of
sodium trifluoroacetate (50 mM) and aglycone (3-FBNH (3 or
6 mg in total volume 50 μL or 100 μL of a 3:1 mixture of
DMSO:PBS) was injected into muscle of three adult 129S-
Gt(ROSA)26Sor/J mice (2 live and 2 post mortem). 19F NMR
spectra were acquired immediately at 4.7 T and every 2 min.
NaTFA served as a chemical shift reference at 0 ppm.
1H MRI T2 Maps of Phantoms of 13 and S-Gal in Agar.
Mixtures of agar and 3,4-cyclohexenoesculetin-β-D-galacto-
pyranoside sodium (S-Gal sodium salt, Sigma-Aldrich Inc., St.
Louis, MO) (5 mM, 40 μL) + ferric ammonium citrate (FAC
2.5 mM, 40 μL) or 13 (5 mM, 40 μL) + FAC (2.5 mM, 40 μL)
with or without β-gal (E801A, 5 units) were prepared in
sections of 384 well plates cut to fit in a 1 turn, 2 cm volume
solenoid coil. T2 maps were acquired at 4.7 T using a spin echo
sequence with variable echo times. MRI parameters were:
FOV = 40 mm × 40 mm, matrix 128 × 128, slice thickness =
1 mm, TR = 6 s, TE = 10, 20, 30, 50, 80, 100, 150, and 200 ms.
Detection of β-Galactosidase by 19F NMR in Cells. Human
MCF7 breast and PC3 prostate cancer cells were transfected to
stably express the E. coli lacZ gene constitutively, as described
previously.8,9 Wild-type and -lacZ cells were grown in culture
dishes under standard conditions and harvested for NMR tests.
19F NMR spectra were acquired at 9.4 T up to 40 h after
addition of 13 (2.11 mg, 5.0 μmol) to MCF7 cells (5.0 × 106)
in 600 μL PBS, and in some cases, Fe3+ (FAC 2.5 μmol) was
added. In addition to evaluating intact cells, additional tests
were conducted with lysed cells. For comparison, equal
numbers of cells were prepared for evaluation intact or follow-
ing lysis. Cell lysis was achieved by a freeze/thaw method: equal
numbers of MCF7-lacZ or PC3-lacZ cells were suspended in
PBS and then frozen at −80 °C for 10 min before thawing at
room temperature over 3 cycles.
RESULTS
■
Each of the target reporter molecules was achieved in high yield
and structures were confirmed by NMR and chemical analyses
(see Supporting Information). The anomeric β-D-configuration
of compounds 4−17 in the 4C1 chair conformation was
confirmed by the observed 1H NMR chemical shifts (δH 4.79−
5.12 ppm) of the anomeric protons and the J1,2 (J ∼8 Hz) and
J2,3 (J ∼10 Hz) coupling constants.29 The anomeric carbon re-
sonances appeared at δC‑1′ 100.05−105.23 ppm in accord with
the β-D-configuration. 19F NMR chemical shifts were measured
with respect to sodium trifluoroacetate (NaTFA, δ = 0 ppm) in
a capillary as an external standard. 12−17 each gave a single
narrow 19F NMR signal between δF −44.0 and −55.0 ppm
(Table 1) essentially invariant (ΔδF ≤ 0.05 ppm) with pH in
the range 3−12 and temperatures from 25 to 37 °C in 0.9%
saline or rabbit whole blood.
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a
Table 1. 19F NMR Chemical Shifts (ppm) of 12−17 and Hydrolytic Rates (μmol/min/unit) of 13−15 with β-gal
No.
12
13
14
15
16
17
δF (substrate)
δF (aglycone)
ΔδF
−54.84
---
−54.62
−62.27
7.65
−54.60
−62.03
7.43
−44.38
−49.61
5.23
−45.02
---
−45.03
---
---
---
---
ν(μmol/min/unit)
---
3.91
1.67
0.55
---
---
a
β-gal (E801A) at 37 °C in PBS (0.1 M, pH = 7.4).
When β-gal (E801A) was added to 12−17 in phosphate
buffered saline (PBS) at 37 °C, only 13−15 were hydrolyzed
releasing the 3- or 5-fluorobenzaldehyde aroylhydrazones ap-
pearing also as single narrow 19F signals shifted upfield between
ΔδF = 5.23−7.65 ppm (δF −49.0 to −63.0 ppm) (Figures 3 and
1
Figure 4. H MRI T2 maps of phantoms of 13 and S-Gal in agar.
Comparison of T2 shortening due to addition of β-gal enzyme to
commercial substrate S-Gal or 13. (A) S-Gal (5 mM) + Fe3+
(2.5 mM); (B) 13 (5 mM) + Fe3+ (2.5 mM); (C) as (A) + β-gal
(E801A, 5 units); (D) as (B) + β-gal (E801A, 5 units).
enhancement to formation of complex between 3-FBNH and
Fe3+. Chemical analysis indicated a 1:2 Fe3+:3-FBNH complex.
When 13 was incubated with MCF7 human breast tumor
cells for 5 h in PBS at 37 °C under 5% CO2 in air, no changes
were observed in the 19F NMR spectrum. Addition of 13 to
stably transfected MCF7-lacZ cells led to cleavage in a smooth
monotonic manner with a rate of 0.25 μmol/min per million
cells, and appearance of a new 19F signal of the released aglycone
3-fluorobenzaldehyde nicotinoyl hydrazone (3-FBNH) around
−62.27 ppm (Figure 5). When ferric ammonium citrate (FAC)
was added after 28 h, the product aglycone 19F signal im-
mediately disappeared, which we attribute to a paramagnetic
relaxation enhancement in the Fe3+:3-FBNH complex.
Given that conversion of substrate appeared much slower in
cells than with enzyme in vitro, tests were conducted to com-
pare intact and lysed cells. Incubation of 13 with equal numbers
of intact or lysed MCF7-lacZ cells showed 25% conversion after
overnight incubation (14 h), whereas lysed cells showed 75%.
Similarly, PC3-lacZ cells showed only 15% conversion, while
lysed PC3-lacZ cells showed 85%.
As an initial proof of principle, 13 and FAC were injected
into wild type (WT) and lacZ-transfected PC3 human prostate
tumor xenografts growing in mice (Figure 6). Spin−lattice re-
laxation time (T1)-weighted images showed a local hyper-
intensity associated with the ferric ions, and a corresponding
drop in the T1 values was observed. For groups of tumors, there
was no significant difference between mean baseline T1 (2.4
0.4 s; n = 2 WT vs 2.4 0.3 s; n = 3 lacZ; p > 0.8). Following
injection of contrast agent (i.e., 13 plus FAC), T1 decreased in
Figure 3. Substrate response to β-galactosidase. Graph shows the loss
□
●
of each of the three agents 13−15 (5.0 μmol; , 13; , 14; Δ, 15)
when incubated with β-galactosidase (E801A, 5 units) in PBS (0.1 M,
600 μL) at 37 °C, as detected by 19F NMR spectroscopy revealing that
13 reacts fastest. When β-galactosidase (20 units) was added to a
solution of 13 (7 mg 16.5 μmol) in 250 μL PBS/DMSO 1:1 at 37 °C,
conversion was detectable by 19F NMR spectroscopy and imaging at
9.4 T (376 MHz: baseline at left and after 2.5 h at right) showing
product 3-fluorobenzaldehyde nicotinoyl hydrazone (3-FBNH).
S1; Table 1), which is comparable to shifts reported previously
for 19F NMR gene reporter molecules.7−9,29,30 The hydrolysis
of 13−15 proceeded smoothly indicating that the liberated
aglycones 3- or 5-fluorobenzaldehyde aroylhydrazones had no
inhibitory effects on β-gal, and the shapes of the kinetic curves
suggest straightforward zero-order kinetics. The rate of reaction
of 13 (ν(E801A) = 3.91 μmol/min/unit) was comparable to that
reported previously for 3-O-(β-D-galactopyranosyl)-6-fluoro-
pyridoxol (GFPOL)30 and much faster than the other
molecules here, so it was chosen for further studies. Enzyme
induced hydrolysis was also observable by 19F MRI (Figure 3).
Titration of product 3-fluorobenzaldehyde nicotinoyl hydra-
zone (3-FBNH) showed a small chemical shift (∼0.5 ppm) in
the pH range 6.5−7.7 (Figure S2).
Proton MRI of agar phantoms of 13 or commercial S-Gal
(3,4-cyclohexenoesculetin β-D-galactopyranoside) with ferric
ions showed very similar T2 (152 21 ms vs 156 25 ms;
Figure 4). Incorporation of β-gal in the agar yielded much re-
duced T2 values and the difference was much greater for 13 than
S-Gal (23 12 ms vs 78 11 ms: alternately, ΔR2 36.9 s−1 vs
6.4 s−1). By analogy with S-Gal,24 we attribute the relaxation
WT tumors (T1 = 1.5
0.4, p < 0.2), but the change was
significant in lacZ tumors (T1 = 1.1 0.4; p < 0.01). For the
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Figure 5. Detection of β-gal activity in cultured cells. 376 MHz 19F
NMR spectra of 13 (2.11 mg, 5.0 μmol) after addition to stably
transfected MCF7-lacZ cells (5.0 × 106). Fe3+ (2.5 μmol) was added
after 28 h. Each spectrum was acquired in 205 s, and enhanced with an
exponential line broadening 40 Hz.
Figure 7. Detection of β-gal activity in vivo by 19F NMR. Left:
Following injection of 13 (1 mg) into a PC3-WT tumor, 19F NMR
spectroscopy showed substrate at −54.6 ppm with respect to sodium
trifluoroacetate reference (lower spectrum). Following injection of
0.6 mg FAC, the substrate remained visible (upper left). Spectra were
acquired in about 2½ min and enhanced with 60 Hz exponential line
broadening prior to Fourier transformation giving a SNR of 35. Right:
Similar injection into PC3-lacZ tumor showed minimal 19F NMR
signal and there was minimal change after addition of FAC (upper).
Upper spectra: A mixture of sodium trifluoroacetate and aglycone
(3-FBNH; 3 mg) in total volume of 50 μL was injected into muscle of
a dead adult 129S-Gt(ROSA)26Sor/J mouse. 19F NMR spectra were
acquired immediately and every 2 min. NaTFA served as a chemical
shift reference at 0 ppm and remained quite constant. Meanwhile
aglycone was initially seen with SNR = 194 at −62.8 ppm, but signal
decreased and was no longer observed after 6 min.
same regions of the tumors, a large signal decrease was ob-
1
served in spin−spin (T2)-weighted H images in PC3-lacZ
tumors only at the injection site. T2 was similar before injection
of contrast agent (T2 = 55 5 ms (lacZ) vs T2 = 52 16 ms
(WT)). Following administration of contrast agent, T2 changed
significantly in lacZ tumors (T2 = 39 5 ms; p < 0.05), but not
in WT tumors (T2 = 53 15 ms; p > 0.9). 19F spectroscopy
showed the presence of substrate 13 in the WT tumors, which
decreased slowly (Figure 7), but no aglycone product. In
corresponding PC3-lacZ tumors, no substrate or product signal
was observed by 19F NMR. Enzyme activity was confirmed
postmortem based on X-gal staining of slices of PC3-lacZ
tumors, which turned intense blue, whereas wild-type tumors
showed no blue color (Figure 6).
Lack of detectable 19F NMR could have been caused by
clearance of the product from lacZ tumors in vivo or potentially
precipitation of the product by association with ferric ions,
Figure 6. Imaging β-gal activity in vivo. In vivo study showing lacZ gene-reporter activity detected by 1H MRI in representative PC3 tumor xenografts
in SCID mice after intratumoral injection (100 μL total volume) of ferric ammonium citrate (FAC) and 13. The presence of FAC + 13 is seen in
T1-weighted images (TR/TE = 300/12 ms) and T1 maps (white arrows) in both wild-type tumors (columns 1 and 2, row 2) and lacZ tumors
(columns 1 and 2, row 4). Baseline tumor T1 = 2.8 0.1 s for lacZ and 2.6 0.2 s for WT, versus 1.1 0.6 s for lacZ and 1.8 0.5 s for WT
following injection of 13 + FAC. Significant change in T2-weighted images (TR/TE = 6000/50 ms, column 3, row 4) and T2 values (column 4, row
4) was seen only in the lacZ-transfected tumors post-injection of FAC and 13 (pink arrows). Baseline T2 = 56 4 ms for lacZ and T2 = 63 7 ms
for WT became T2 = 34 10 ms for lacZ and T2 = 63 18 ms for WT. Green arrow indicates anomalous injection outside tumor, and this region
was excluded from analysis. Histological sections at right (original magnification × 100) confirm intense β-gal expression in lacZ tumor based on
X-gal and H&E staining (blue, lower slide) with essentially no activity in WT tumor (pink, upper slide).
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rendering it NMR invisible. To gain further insight into the lack
of 19F signal, product aglycone was injected into muscle of
ROSA26 mice, which express lacZ throughout their bodies.
Internal NaTFA standard and aglycone were immediately
detectable, but over a few minutes, the aglycone signal at
−61.5 ppm disappeared in both live and dead mice, while TFA
remained visible in the dead animal (Figure 7).
19F NMR is gaining popularity as a reporter for diverse
enzyme reactions based on various strategies. The simplest is
change in chemical shift that we and others have exploited
extensively.8,9,34,35 More recently, differential relaxation rates
have been exploited based on paramagnetic relaxation enhance-
ment (PRE)10,36 or differential restricted mobility37,38 to reveal
enzyme activity based on changes in line broadening. In addi-
tion, several recent reports have presented bimodal reporter
strategies revealing enzyme activity based on 19F NMR and
fluorescence.38,39
Ultimately, we hope to develop this approach for systemic
delivery of reporter molecules, but direct injection into the
tissues of interest already demonstrates selective detection of
lacZ expression versus WT. The multimodality approach re-
presents a new paradigm exploiting 19F NMR together with
both T1 and T2 1H MRI contrast to reveal the presence or
absence of enzyme activity. The observations are in accord with
renewed excitement and innovations in 19F NMR notably for
detecting enzyme activity.37,39−42
DISCUSSION
■
We have demonstrated a novel dual 19F/1H MR lacZ gene
detection approach by introducing a fluorine atom into iron-
chelating aroylhydrazone aglycones of β-D-galactopyranosides.
When activated by β-gal, the released fluoroaroylhydrazones
display a distinct chemical shift with respect to the substrate
and can be spontaneously trapped by Fe3+ at the site of enzyme
activity forming highly relaxing complexes in situ. The com-
plexes cause strong T2 relaxation, providing 1H MRI contrast to
reveal lacZ expression.
We believe the T1 contrast following injection is attributable
to free ferric ions from FAC and this was observed in both WT
and lacZ tumors (Figure 6). However, β-gal activity was clearly
revealed by the colocalized hypointensity in T2-weighted
images and corresponding significant shortening in T2, whereas
no T2 contrast was observed for WT tumors.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental details and molecular characterization. This
material is available free of charge via the Internet at http://
pubs.acs.org.
The utility of a reporter molecule is predicated on a re-
asonable rate of activity. The conversion rates in vitro were very
slow (Figure 3). Aglycone was released much more rapidly by
lysed MCF7-lacZ or PC3-lacZ cells indicating that cell
permeation is a strong barrier to activity. However, contrast
was observed almost immediately in lacZ tumors upon direct
intratumoral injection. Such differential rates of activity have
also been reported for 19F reporters.8,9 The ultimate goal is
development of reporter molecules which may be administered
systemically. At this stage, the method requires direct intra-
tumoral injection, but we note that this limitation has also been
encountered by most other reports regarding reporter
molecules for β-gal using NMR, radionuclides, or photoacoustic
tomography.8,9,12,16,21,24 To date, systemic administration has
generally been restricted to optical imaging approaches14,18,31
and a single report regarding a Gd-based contrast agent.6
Ideally, 19F NMR would show both substrate and product,
and this was observed in cells (Figure 5). However, the liberat-
ed product signal was absent in the presence of ferric ions and
in vivo. This could be attributed to clearance of the product
from lacZ tumors and ROSA26 muscle in vivo, but the post-
mortem test in the ROSA26 mouse also indicated signal dis-
appearance over a period of 6 min consistent with precipitation
of the product (possibly in association with endogenous ferric
ions), which renders it NMR invisible. 19F signal for TFA
remained visible over this time. The 19F NMR is able to locate
substrate, but not product, and we are seeking alternate mol-
ecules, which can reveal both substrate and product.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (214)-648-8926. Fax: (214)-648-4538. E-mail: Ralph.
Mason@UTSouthwestern.edu.
Present Address
†Currently at the School of Biological and Health Systems
Engineering, Arizona State University, Tempe, AZ
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Supported in part by NCI R21 CA120774, DOD Breast Cancer
Initiative IDEA award DAMD17-03-1-0343, and the Small
Animal Imaging Research Program, which is supported in part
by NCI U24 CA126608 and P30 CA142543. NMR experi-
ments were conducted at the Advanced Imaging Research
Center, an NIH BTRP facility (P41-RR02584). We are grateful
to Praveen K. Gulaka, Jennifer Magnusson, Jennifer McAnally,
and Ya Ren for expert technical assistance.
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