An activity-based near-infrared glucuronide trapping probe for imaging β-glucuronidase expression in deep tissues

Article abstract of DOI:10.1021/ja209335z

β-glucuronidase is an attractive reporter and prodrug-converting enzyme. The development of near-IR (NIR) probes for imaging of β-glucuronidase activity would be ideal to allow estimation of reporter expression and for personalized glucuronide prodrug cancer therapy in preclinical studies. However, NIR glucuronide probes are not yet available. In this work, we developed two fluorescent probes for detection of β-glucuronidase activity, one for the NIR range (containing IR-820 dye) and the other for the visible range [containing fluorescein isothiocyanate (FITC)], by utilizing a difluoromethylphenol-glucuronide moiety (TrapG) to trap the fluorochromes in the vicinity of the active enzyme. β-glucuronidase- mediated hydrolysis of the glucuronyl bond of TrapG generates a highly reactive alkylating group that facilitates the attachment of the fluorochrome to nucleophilic moieties located near β-glucuronidase-expressing sites. FITC-TrapG was selectively trapped on purified β-glucuronidase or β-glucuronidase-expressing CT26 cells (CT26/mβG) but not on bovine serum albumin or non-β-glucuronidase-expressing CT26 cells used as controls. β-glucuronidase-activated FITC-TrapG did not interfere with β-glucuronidase activity and could label bystander proteins near β-glucuronidase. Both FITC-TrapG and NIR-TrapG specifically imaged subcutaneous CT26/mβG tumors, but only NIR-TrapG could image CT26/mβG tumors transplanted deep in the liver. Thus NIR-TrapG may provide a valuable tool for visualizing β-glucuronidase activity in vivo.

Full text of DOI:10.1021/ja209335z

Article
pubs.acs.org/JACS
An Activity-Based Near-Infrared Glucuronide Trapping Probe for
Imaging β-Glucuronidase Expression in Deep Tissues
Ta-Chun Cheng,†,◆ Steve R. Roffler,‡,◆ Shey-Cherng Tzou,§ Kuo-Hsiang Chuang,§ Yu-Cheng Su,‡,∥
Chih-Hung Chuang,⊥ Chien-Han Kao,† Chien-Shu Chen,# I-Hong Harn,§ Kuan-Yi Liu,○
Tian-Lu Cheng,*,§,∇ and Yu-Ling Leu*,○
†Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
‡Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
§Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan
∥Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan
⊥Institutes of Basic Medical Sciences, National Cheng Kung University, Tainan, Taiwan
#School of Pharmacy, China Medical University, Taichung, Taiwan
∇Cancer Center, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
○Department of Pharmacy, Chia Nan University of Pharmacy and Science, Tainan, Taiwan
S
* Supporting Information
ABSTRACT: β-glucuronidase is an attractive reporter and
prodrug-converting enzyme. The development of near-IR
(NIR) probes for imaging of β-glucuronidase activity would
be ideal to allow estimation of reporter expression and for
personalized glucuronide prodrug cancer therapy in preclinical
studies. However, NIR glucuronide probes are not yet available.
In this work, we developed two fluorescent probes for detection
of β-glucuronidase activity, one for the NIR range (containing
IR-820 dye) and the other for the visible range [containing
fluorescein isothiocyanate (FITC)], by utilizing a difluorome-
thylphenol−glucuronide moiety (TrapG) to trap the fluorochromes in the vicinity of the active enzyme. β-glucuronidase-
mediated hydrolysis of the glucuronyl bond of TrapG generates a highly reactive alkylating group that facilitates the attachment
of the fluorochrome to nucleophilic moieties located near β-glucuronidase-expressing sites. FITC-TrapG was selectively trapped
on purified β-glucuronidase or β-glucuronidase-expressing CT26 cells (CT26/mβG) but not on bovine serum albumin or non-β-
glucuronidase-expressing CT26 cells used as controls. β-glucuronidase-activated FITC-TrapG did not interfere with β-
glucuronidase activity and could label bystander proteins near β-glucuronidase. Both FITC-TrapG and NIR-TrapG specifically
imaged subcutaneous CT26/mβG tumors, but only NIR-TrapG could image CT26/mβG tumors transplanted deep in the liver.
Thus NIR-TrapG may provide a valuable tool for visualizing β-glucuronidase activity in vivo.
di-β-D-glucuronide (FDGlcU)9 and 4-methylumbelliferyl β-D-
INTRODUCTION
■
glucuronide (MU-GlcA)14 have been developed to increase the
detection sensitivity. Our previous study demonstrated that
FDGlcU can be used to determine βG activity both in vitro and
in vivo.9 The penetrability of FDGlcU, however, is insufficient
to image βG-expressing tumors in deep organs in living
animals. Moreover, the signal generated with the FDGlcU
probe rapidly diffuses away from βG-expressing sites and
therefore only allows imaging of subcutaneous βG-expressing
tumors over a relatively narrow time window.9 Thus, the
development of an activity-based near-IR (NIR) fluorescent
glucuronide probe (λem > 800 nm) may facilitate imaging of
deep tissues and improve the imaging resolution for βG.
β-glucuronidase (βG) is a lysosomal enzyme1,2 that has been
used as a tumor marker,2−4 a prodrug-converting enzyme for
cancer prodrug therapies,5,6 and a reporter gene for tracking the
location of gene delivery vectors in preclinical studies.7−10 The
ability to image βG activity in vivo would greatly improve βG-
based applications. However, most βG probes are suitable only
for in vitro studies and are not yet available for in vivo imaging
of βG activity. For example, naphthol AS-BI β-D-glucuro-
nide,11,12 p-nitrophenyl-β-D-glucuronide (p-NPG),13 and 5-
bromo-4-chloro-3-indolyl-β-D-glucuronic acid (X-GlcA, X-
Gluc)14,15 are commonly used to detect βG activity in cultured
cells and histological sections, but these colorimetric substrates
have poor sensitivities and relatively narrow dynamic ranges
and hence are not useful in vivo. In an attempt to overcome this
problem, a range of fluorogenic substrates, such as fluorescein
Received: October 4, 2011
Published: January 12, 2012
© 2012 American Chemical Society
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In this study, we developed NIR-TrapG, an activity-based
NIR-fluorescent difluoromethylphenol−glucuronide probe.
βG-mediated hydrolysis of the glucuronyl bond generates a
highly reactive quinine methide intermediate that facilitates the
attachment of the fluorochrome to nucleophilic side chains near
βG-expressing sites (Figure 1). The NIR-TrapG probe is
examined the specificity of these trapping probes by incubating
FITC-TrapG with recombinant Escherichia coli βG (eβG) or
βG-expressing mouse colon cancer cells (CT26/mβG) in vitro.
We investigated whether alkylation by activated FITC-TrapG
affects the βG activity and whether FITC-TrapG could label
bystander proteins in the vicinity of βG activity. Finally, we
examined whether NIR-TrapG or FITC-TrapG could specif-
ically image the location of βG-expressing CT26 tumors
transplanted under the skin or deep in the livers of mice. NIR-
TrapG displayed high tissue penetrability and thus may be a
useful tool for tracking βG activity in vivo and for optimizing
preclinical βG-based therapies and imaging systems.
RESULTS
■
Development of Glucuronide Trapping Probes. To
develop novel glucuronide trapping probes, fluorescein
isothiocyanate (FITC) and the dye IR-820 were linked to a
glucuronide group via a difluoromethylphenol trapping moiety
to form FITC-TrapG and NIR-TrapG, respectively. The
glucuronide group acts as a hydrophilic and cell-impermeable
βG substrate. βG-mediated hydrolysis of the glucuronyl bond
in the probe exposes the quinine methide group, which can
cross-link FITC or IR-820 to nearby nucleophiles (Figure 1).
The design and synthesis of FITC-TrapG and NIR-TrapG are
shown in Schemes 1−5 (see the Experimental Section). Details
of the syntheses are described in the Experimental Section and
the Supporting Information.
Figure 1. Mechanism of the βG activity-based trapping probe. βG-
mediated hydrolysis of the glucuronyl bond generates a highly reactive
quinine methide intermediate that leads to cross-linking of the probe
to nearby nucleophiles.
advantageous in that it provides a specific and direct “activity-
based” enzyme profile for βG. In addition, the high
penetrability of NIR signals could make this strategy useful
for deep-tissue imaging.16−18 Furthermore, activity-based
probes have been developed for in vivo profiling of enzymatic
activities.19−22
Characterization of Glucuronide Trapping Probes in
Vitro. To examine whether βG can specifically activate FITC-
TrapG and expose the reactive alkylating group in vitro,
increasing amounts of FITC-TrapG were incubated with either
purified eβG or bovine serum albumin (BSA) adsorbed in
Here we report the design and synthesis of two fluorescent
probes for βG: NIR-TrapG and FITC-TrapG. We first
■
□
Figure 2. βG-specific activation of FITC-TrapG in vitro. Increasing amounts of FITC-TrapG were incubated with (A) βG ( ) or BSA ( ) or (B)
■
□
CT26/mβG ( ) or CT26 ( ) precoated on the wells of a microtiter plate. Activation and trapping of FITC-TrapG was determined by ELISA using
an anti-FITC antibody. After FITC-TrapG treatment, the βG activity was determined by hydrolysis of the p-NPG substrate in (C) purified proteins
(eβG, BSA) or (D) cells (CT26/mβG, CT26).
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microtiter plates. After excess probe was washed away, FITC
attached to proteins in the wells was detected by enzyme-linked
immunosorbent assay (ELISA) using an anti-FITC antibody.
Figure 2A shows that the absorbance at 405 nm increased with
increasing concentrations of the probe added to eβG.
Conversely, incubation of FITC-TrapG with BSA did not
result in accumulation of FITC in the wells. Similarly, mouse
βG (mβG) expressed on the surface of CT26 cells activated
FITC-TrapG and showed trapping of FITC (Figure 2B). No
color development was noted after the addition of FITC-TrapG
to parental CT26 cells. These results indicate that the
glucuronide trapping probes were specifically activated by βG
and stably retained. Furthermore, activated FITC-TrapG did
not hamper βG activity, as 100% enzymatic activity was
maintained even at the highest concentration of FITC-TrapG
(40 μg/mL) (Figure 2C,D). We conclude that activated FITC-
TrapG can be retained at sites of βG activity without affecting
its enzymatic activity.
4B). Similarly, NIR-TrapG could specifically label βG-
expressing cells in vitro and subcutaneous βG-expressing
tumors in vivo (Figure 5). The cell-associated NIR intensity
was 2.4, 2.6, and 2.8 times greater in CT26/mβG cells than in
the control CT26 cells upon pretreatment with 0.5, 1, and 2
μg/mL NIR-TrapG, respectively (Figure 5A). The NIR
fluorescence signal was 4.25, 4.92, and 5.21 times greater in
subcutaneous CT26/mβG tumors than in the control CT26
tumors at 24, 48, and 72 h, respectively, after probe injection
(Figure 5B). Similar results were confirmed in a biodistribution
study of FITC-TrapG and NIR-TrapG (Figure S1 in the
Supporting Information). However, some nonspecific signals
were detected in the liver, kidney, bladder, and intestines. This
result suggests that the glucuronide trapping probe was
eliminated by both the urinary and hepatobiliary routes. We
conclude that the glucuronide trapping probes can image βG
activity in live animals.
Imaging of βG Activity in Tumors Transplanted in
Livers. Deep tissues present a major technical challenge to
optical imaging. To test whether NIR-TrapG can be used for
deep-tissue imaging, we injected NIR-TrapG to BALB/c nude
mice that had CT26 or CT26/mβG tumors transplanted under
their liver capsules. FITC-TrapG was also injected in separate
mice with liver tumors. Figure 6A shows that NIR signals were
detected in live mice bearing CT26/mβG tumors in their livers.
Consistent with the noninvasive imaging results, significantly
stronger NIR signals were recorded in livers isolated from mice
bearing CT26/mβG tumor transplants than in mice bearing
control CT26 tumors. On the contrary, no FITC signal was
detected in live mice; FITC signals were only detected when
animals were killed and livers were placed directly under the
detector (Figure 6B). Collectively, these results demonstrate
that NIR-TrapG can detect βG activity in deep tissues.
Conceptually, the activated FITC-TrapG probe can alkylate
any nucleophile in close proximity to βG. To test whether the
activated probes could label bystander nucleophiles, we added
FITC-TrapG to a mixture of eβG and BSA in solution.
Addition of FITC-TrapG resulted in labeling of FITC groups
on both eβG (74 kDa) and BSA (66 kDa) (Figure 3). In the
DISCUSSION
■
We have developed an NIR glucuronide trapping probe (NIR-
TrapG) for in vivo imaging of βG expression in deep tissues.
βG-mediated hydrolysis of the glucuronyl bond of NIR-TrapG
leads to cross-linking of the probe onto nearby nucleophiles at
βG-expressing sites. The in vitro analyses demonstrated that
this novel glucuronide trapping probe does not affect βG
activity and that the activated probe can label bystander
proteins. The high penetrability of NIR signals through tissues
renders this probe especially useful for noninvasive optical
imaging of βG expression in deep tissues.
Figure 3. Bystander trapping by FITC-TrapG. FITC-TrapG was
incubated with eβG, BSA, or a mixture of eβG and BSA. Activation
and trapping of FITC-TrapG on proteins was detected by western
blotting using an anti-FITC antibody (right panel). Protein loading
was visualized by Coomassie blue staining (left panel).
NIR-fluorescent dyes are useful for biomedical studies. NIR
fluorochromes (λem = 700−900 nm) have high signal/
background ratios and penetrate tissues better than those
with shorter wavelengths.23−25 A β-galactosidase (LacZ)-
activated far-red probe (DDOAG, λem = 659 nm) that
facilitates noninvasive monitoring of the expression and activity
of LacZ in vivo has been reported.26 Moreover, a PEGylated
NIR probe (PEG-NIR797, λem = 797 nm) can detect lung
metastasis in vivo.27 Adams and colleagues also reported that
epidermal growth factor (EGF) conjugated with an NIR dye
(λem = 800 nm) produced better images of EGF-receptor-
positive tumors than Cy5.5 (λem = 710 nm).28 In this report, we
have demonstrated that NIR-TrapG (λem = 820 nm), but not
FITC-TrapG, can be used to visualize βG-expressing tumors in
deep tissues (liver).
absence of βG-mediated activation, FITC-TrapG did not label
the control protein BSA. We conclude that activated FITC-
TrapG can label bystander nucleophiles in the vicinity of βG
enzyme activity.
Imaging of βG Activity in Subcutaneous Tumors. To
examine whether the glucuronide trapping probes can
specifically detect βG activity in vivo, we intravenously injected
FITC-TrapG into BALB/c nude mice bearing subcutaneous
CT26 or CT26/mβG tumors. Fluorescent signals were
measured in live mice with a noninvasive optical imaging
system. Figure 4A shows that the fluorescence intensity
(defined as photons s−1 cm−2 sr−1) in the CT26/mβG tumors
was 4.59, 3.86, and 2.86 times greater than that in the control
CT26 tumors at 24, 48, and 72 h, respectively. In line with the
imaging results, FITC-derived fluorescence was retained in
CT26/mβG tumors but not the control CT26 tumors, which
was consistent with X-GlcA staining for βG activity (Figure
Activity-based probes have gained marked success for in vivo
profiling of enzymatic activities, including proteases,25,29
thymidine kinases,30,31 and galactosidases.26,32 This concept
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Figure 4. Specific activation of FITC-TrapG in vivo. FITC-TrapG was intravenously injected into BALB/c nude mice bearing CT26/mβG (right
flank) and CT26 (left flank) tumors. (A) In vivo optical imaging of FITC-TrapG at 24, 48, and 72 h after probe injection. Tumor tissue was also
harvested to confirm specific fluorescent signals. (B) CT26/mβG and CT26 tumors were resected at 24 h after FITC-TrapG injection, stained with
X-GlcA, and examined under bright-field and fluorescent field illumination. Scale bar: 100 μm.
Figure 5. Optical imaging using NIR-TrapG. (A) Optical imaging of CT26/mβG or CT26 cells (3 × 106/well) pretreated with 2, 1, 0.5, or 0 μg/mL
of NIR-TrapG. (B) Optical imaging of subcutaneous CT26/mβG and CT26 tumors in mice at 24, 48, and 72 h after probe injection.
Figure 6. Deep tissue imaging of βG-expressing tumors using NIR-TrapG. NIR-TrapG or FITC-TrapG was injected into BALB/c mice bearing
CT26 or CT26/mβG tumor transplants in their livers. Noninvasive optical imaging of (A) NIR-TrapG or (B) FITC-TrapG was performed at 24 h
after probe injection. Livers were harvested to confirm the location of the tumors and the specificity of the fluorescent signal. Red dotted lines
indicate tumor locations in the liver.
depends heavily on the specific binding of the probe to the
active site of the target enzyme. Activity-based probes can
irreversibly inactivate the targeted enzymes, as observed for
cysteine protease,33,34 cytosolic 2 class I aldehyde dehydrogen-
ase (cALDH-I),35 transcriptional activator (LasR),36 and
cytochrome P450.37 In contrast, enzymatic activation of
noninhibitory probes leads to signal amplification and thus
improves detection.33 In addition, noninhibitory imaging
probes do not interfere with therapeutic efficiency of enzyme-
based prodrug therapies.25,38 Difluoromethylphenol substrates
have been demonstrated to display trapping activity after
activation by galactosidases39 and sialidases40 but do not
irreversibly inactivate enzymes.41 Our glucuronide trapping
probe does not appear to inhibit the βG activity, which would
allow enhanced activation of the substrate and therefore
improved imaging. Because many glucuronide antitumor
prodrugs have been developed, including 9-aminocamptothecin
glucuronide (9ACG)42 and p-hydroxyaniline mustard glucur-
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Scheme 1. Chemical Structure and Synthesis of Methyl 1-O-(2-Difluoromethyl-4-aminophenyl)-β-D-glucopyranuronate (7)
Scheme 2. Formation and Autodegradation of N′-Fluorescein-N″-[4-O-(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranuronate)-3-
difluoromethylphenyl]thiourea (8)
onide (BHAMG),43 to treat selectively cancers with βG
overexpression,4,42 the glucuronide trapping probes may be
useful for (1) screening tumors with βG overexpression and (2)
monitoring the efficacy of glucuronide prodrugs in personalized
anticancer therapy.
developing a variety of novel imaging probes to detect other
enzymes relevant to human diseases.
CONCLUSIONS
■
The NIR glucuronide trapping probe possesses several
attractive attributes. The probe has high penetrability for
noninvasive imaging of βG activity in deep tissues. The
trapping moiety (difluoromethylphenol) does not inhibit βG
activity, allowing enhancement of the image intensity. On the
basis of these advantages, we believe that the glucuronide
trapping probe may be a valuable tool for imaging βG activity in
preclinical studies. The trapping strategy may be extended to
other imaging systems or other enzymes.
The trapping moiety (difluoromethylphenol) may be a
versatile linker for multiple imaging systems because, at least
conceptually, difluoromethylphenol could be coupled to other
imaging probes to detect βG activity. In this report, we have
demonstrated that difluoromethylphenol can be conjugated to
two fluorescent probes (FITC and IR-820) for optical imaging.
Following derivative chemical principles, difluoromethylphenol
could be linked to a glucuronide group with different imaging
agents, such as 1,4,7-tricarboxymethylene-1,4,7,10-tetraazacy-
clododecane (DO3A)44 and 1,4,7,10-tetraazacyclododecane-
N,N′,N″,N‴-tetraacetic acid (DOTA)25 for magnetic resonance
imaging or radioiodinated tyramine4 and fluoroethylamine45 for
nuclear imaging [positron emission tomography (PET) and
single-photon emission computed tomography (SPECT)]. The
development of βG-specific probes may further broaden the
selection of tools for monitoring βG expression in vivo.
Furthermore, the trapping strategy may hold great potential for
EXPERIMENTAL SECTION
■
Design and Synthesis of FITC-TrapG and NIR-TrapG. The
detailed chemistry procedures are described in the Supporting
Information. To develop novel glucuronide trapping probes, FITC
and IR-820 were linked to a glucuronide group by a trapping moiety,
difluoromethylphenol, to form the activity-based probes FITC-TrapG
and NIR-TrapG, respectively. FITC-TrapG and NIR-TrapG were
prepared from methyl 1-α-bromo-1-deoxy-2,3,4-tri-O-acetyl-β-D-glu-
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Scheme 3. Chemical Structure and Synthesis of N′-Fluorescein-N″-[4-O-(β-D-glucopyranuronic acid)-3-difluoromethylphenyl]-
S-methylthiourea (FITC-TrapG, 10)
Scheme 4. Chemical Structure and Synthesis of IR-820-Containing Isothiocyanate 13
copyranuronate (3) and 2-hydroxy-5-nitrobenzaldehyde using a
previously published method for the synthesis of glucuronide
derivatives46 with minor modifications. The aldehyde group in 4 was
fluorinated to a difluoromethyl group with diethylaminosulfur
trifluoride (DAST). The nitro group in 5 was reduced in a
palladium-catalyzed reaction to obtain 6 (Scheme 1). For the synthesis
of FITC-TrapG, the aniline in 6 was reacted with FITC to obtain
methyl O-acetyl-protected FITC-TrapG (8) (Scheme 2). However,
when compound 8 was deprotected with sodium methoxide, the
thiourea linkage was hydrolyzed, indicating that the thiourea linkage is
susceptible to hydrolysis at basic or physiological pH. Thus, we
modified the synthesis pathway to enhance the stability. The acetyl
group in compound 6 was deprotected by sodium methoxide to give 7
(Scheme 1). The aniline in 7 was condensed with FITC, after which
methyl iodide (CH3I) was used to create an S-methyl pseudothiourea
linkage,47 affording methyl-protected FITC-TrapG (9) (Scheme 3).
Next, 9 was deprotected with potassium trimethylsilanolate, after
which acidification with 1 N HCl afforded FITC-TrapG (10).
chlorocyanine dye was condensed with p-nitrothiophenol by
nucleophilic substitution to obtain compound 11 (Scheme 4). The
nitro group in 11 was reduced using SnCl2·2H2O to generate an
aminothioether dye, compound 12. The amino group in 12 was
reacted with thiophosgene to generate the isothiocyanate, compound
13. NIR-TrapG was then prepared by the reaction of 7 with 13
followed by addition of CH3I to create the S-methyl pseudothiourea
linkage of methyl-protected NIR-TrapG (14) (Scheme 5). Next,
compound 14 was deprotected with potassium trimethylsilanolate,
after which acidification with 1 N HCl afforded NIR-TrapG (15).
Cells and Mice. CT26 and CT26/mβG cells9 were maintained in
Dulbecco’s minimal essential medium (DMEM) (Sigma-Aldrich, St
Louis, MO) supplemented with 10% bovine calf serum, 100 μg/mL
penicillin, and 100 μg/mL streptomycin at 37 °C in an atmosphere of
5% CO2. Female BALB/c nude mice (BALB/cAnN.Cg-Foxn1nu/
CrlNarl), 5−6 weeks old, were purchased from the National
Laboratory Animal Center, Taiwan. The animal experiments were
conducted in accordance with the standards set forth by the Kaohsiung
Medical University Institutional Animal Care and Use Committee.
For the synthesis of NIR-TrapG, the fluorescein group in FITC-
TrapG was replaced by an NIR dye (IR-820). The commercial IR-820
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Scheme 5. Chemical Structure and Synthesis of the N′-(p-Aminophenyl)thioether of IR-820-N″-[4-O-(β-D-Glucopyranuronic
acid)-3-difluoromethylphenyl]-S-methylthiourea (NIR-TrapG, 15)
Specific Activation of FITC-TrapG in Vitro. Purified eβG (5 μg/
well) and BSA (5 μg/well) (Sigma-Aldrich) were coated overnight in
96-well microtiter plates in phosphate-buffered saline (PBS) (pH 8.0)
and then blocked with 5% skim milk at room temperature for 2 h.
CT26/mβG and control CT26 cells were seeded at 1.5 × 105 cells/
well in 96-well microtiter plates in culture medium overnight. FITC-
TrapG was 2-fold serially diluted (starting from 40 μg/mL) in PBS
and then added to the proteins or cells at 37 °C for 1 h. The plates
were washed with PBS and then stained with a mouse anti-FITC
antibody (Sigma-Aldrich) followed by a horseradish peroxidase-
conjugated goat anti-mouse immunoglobulin G (IgG) antibody
(Jackson ImmunoResearch Laboratories, West Grove, PA). Following
three washes with PBS, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS) (Sigma-Aldrich) with 0.03% H2O2 (Sigma-Aldrich) was
added to the wells, and color development was measured on a
microplate reader (Molecular Devices, Menlo Park, CA) at OD 405
nm.
βG Enzymatic Activity after FITC-TrapG Treatment. To test
whether activated FITC-TrapG inactivates βG enzyme activity, plate-
bound 5 μg/well eβG or 1.5 × 105 cells/well CT26/mβG cells were
incubated with 40−0 μg/mL FITC-TrapG (2-fold serially diluted) as
described above. Following proper washing in PBS, 5 mM p-NPG
(Sigma-Aldrich) was added to the plates. Color development was
measured on a microplate reader at OD 405 nm.
Bystander Trapping of FITC-TrapG. Purified eβG, BSA, or a
mixture of eβG/BSA (50 μg) was incubated with FITC-TrapG (40
μg/mL) at 37 °C for 1 h. Proteins were precipitated using cold
acetone, centrifuged at 12000g for 15 min, electrophoresed on a 10%
reducing SDS-PAGE (0.5 μg/lane), and transferred to a nitrocellulose
membrane (Pall, Port Washington, NY). Membranes were blocked
with 5% skim milk in PBS. FITC attached to proteins was detected by
immunoblotting using a mouse anti-FITC antibody (Sigma-Aldrich), a
horseradish peroxidase-conjugated goat anti-mouse IgG antibody
(Jackson ImmunoResearch Laboratories), and an enhanced chem-
iluminescence kit (Millipore, Billerica, MA).
In Vivo Imaging of FITC-TrapG and NIR-TrapG. BALB/c nude
mice (n = 3) bearing CT26 (in left flanks) and CT26/mβG (in right
flanks) tumors (50−100 mm3) were intravenously injected with 500
μg of FITC-TrapG/mouse or 100 μg of NIR-TrapG/mouse in 100 μL
of PBS. The fluorescence intensity in the tumors and other internal
organs was recorded on an IVIS 50 optical imaging system (Caliper
Life Sciences, Hopkinton, MA) at 24, 48, and 72 h after probe
injection. We also tested 100 μg of FITC-TrapG/mouse, but there was
no significant difference (data not shown). The λex/λem were 490 nm/
525 nm and 710 nm/835 nm for FITC-TrapG and NIR-TrapG,
respectively. The acquisition time was 10 s for both probes. We
calculated the ratio of fluorescence intensities for the βG-expressing
and control tumors as (CT26/mβG − background)/(CT26 −
background).
In Vitro Imaging of NIR-TrapG. CT26 and CT26/mβG cells (3
× 106) were stained with 2, 1, 0.5, or 0 μg of NIR-TrapG/mL in PBS
containing 0.05% BSA at 37 °C for 2 h. The cells were washed with
PBS three times, and the fluorescence was detected on an IVIS 50
optical imaging system.
Histological Analysis. Tumors were excised at 24 h after FITC-
TrapG injection, embedded in OCT compound (Tissue-Tek,
Torrance, CA) at −80 °C, and sectioned into 10 μm slices. Adjacent
tumor sections were stained for βG activity with the β-glucuronidase
Reporter Gene Staining Kit (Sigma-Aldrich). The sections were
viewed in bright-field and fluorescence modes on an Eclipse TE2000-
U inverted microscope (Nikon, Tokyo, Japan).
Tumor Transplantation into Liver and Tumoral Imaging
Using FITC-TrapG or NIR-TrapG. After BALB/c nude mice were
anesthetized with ketamine/xylazine (135 mg/kg; 15 mg/kg), the
abdomenal cavities were surgically opened, and 0.1 cm × 0.2 cm pieces
of CT26/βG or CT26 tumors were transplanted under the capsule of
the livers (n = 3 for each group). One week after the transplantation,
mice were intravenously injected with 500 μg of FITC-TrapG/mouse
or 100 μg of NIR-TrapG/mouse in 100 μL of PBS. Whole-body
fluorescent signals were recorded on an IVIS 50 optical imaging
system at 24 h after probe injection. After noninvasive imaging, the
livers were harvested and placed directly under the detector of the
IVIS 50 system to reaffirm the location of the tumors.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed chemistry procedures, NMR data, and biodistribution
of FITC-TrapG and NIR-TrapG. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
tlcheng@kmu.edu.tw.; yulin@mail.chna.edu.tw
Author Contributions
◆These authors contributed equally.
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dx.doi.org/10.1021/ja209335z | J. Am. Chem. Soc. 2012, 134, 3103−3110

Journal of the American Chemical Society
Article
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ACKNOWLEDGMENTS
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This work was supported by grants from the National Research
Program for Biopharmaceuticals, National Science Council,
Taipei, Taiwan (NSC 100-2325-B-037-001, 100-2320-B-041-
002, NSC98-2320-B-041-003), Academia Sinica (AS-98-TP-
B09), the Department of Health, Executive Yuan, Taiwan
(DOH100-TD-N-111-010, DOH100-TD-C-111-002), and
Kaohsiung Medical University Research Foundation
(KMUER014).
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