Design strategy for a near-infrared fluorescence probe for matrix metalloproteinase utilizing highly cell permeable boron dipyrromethene

Article abstract of DOI:10.1021/ja303931b

Near-infrared (NIR) fluorescence probes are especially useful for simple and noninvasive in vivo imaging inside the body because of low autofluorescence and high tissue transparency in the NIR region compared with other wavelength regions. However, existi

Full text of DOI:10.1021/ja303931b

Article
pubs.acs.org/JACS
Design Strategy for a Near-Infrared Fluorescence Probe for Matrix
Metalloproteinase Utilizing Highly Cell Permeable Boron
Dipyrromethene
Takuya Myochin, Kenjiro Hanaoka, Toru Komatsu, Takuya Terai, and Tetsuo Nagano*
Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
S
* Supporting Information
ABSTRACT: Near-infrared (NIR) fluorescence probes are especially
useful for simple and noninvasive in vivo imaging inside the body because
of low autofluorescence and high tissue transparency in the NIR region
compared with other wavelength regions. However, existing NIR
fluorescence probes for matrix metalloproteinases (MMPs), which are
tumor, atherosclerosis, and inflammation markers, have various
disadvantages, especially as regards sensitivity. Here, we report a novel
design strategy to obtain a NIR fluorescence probe that is rapidly
internalized by free diffusion and well retained intracellularly after
activation by extracellular MMPs. We designed and synthesized four candidate probes, each consisting of a cell permeable or
nonpermeable NIR fluorescent dye as a Forster resonance energy transfer (FRET) donor linked to the NIR dark quencher BHQ-
̈
3 as a FRET acceptor via a MMP substrate peptide. We applied these probes for detection of the MMP activity of cultured HT-
1080 cells, which express MMP2 and MT1-MMP, by fluorescence microscopy. Among them, the probe incorporating
BODIPY650/665, BODIPY-MMP, clearly visualized the MMP activity as an increment of fluorescence inside the cells. We then
applied this probe to a mouse xenograft tumor model prepared with HT-1080 cells. Following intratumoral injection of the
probe, MMP activity could be visualized for much longer with BODIPY-MMP than with the probe containing SulfoCy5, which
is cell impermeable and consequently readily washed out of the tissue. This simple design strategy should be applicable to
develop a range of sensitive, rapidly responsive NIR fluorescence probes not only for MMP activity, but also for other proteases.
MMP is highly water soluble and cell impermeable.8
INTRODUCTION
■
Consequently, the activated probes are rapidly washed out
from the tissue. To overcome this problem, Tsien et al.
developed a probe utilizing a cell-penetrating peptide,9 which
enables the MMP-activated probe to penetrate into cells, where
it is retained for a relatively long period. However, this probe
suffers from toxicity of the cell-penetrating peptide and slow
uptake into cells,10 which occurs via endocytosis or macro-
pinocytosis.11 Herein, we propose a simple design approach to
obtain a highly sensitive fluorescence probe for MMP by
utilizing NIR fluorescent dyes that exhibit high cell permeability
via rapid free diffusion and good intracellular retention after
activation (Figure 1), thereby providing a well-sustained
fluorescence signal with very high signal-to-noise ratio.
Proteases are involved in the regulation of diverse disease
processes and are considered to have potential clinical value
both as biomarkers and therapeutic targets. Among them,
matrix metalloproteinases (MMPs) are a family of zinc
metalloproteinases, which play important roles in degrading
the extracellular matrix (ECM) and in both physiological and
pathological processes of tissue remodeling.1 MMPs activity is
also considered to be a marker for embryogenesis, wound
healing, inflammation, arthritis, cardiovascular disease, and
cancer.2 For example, MMP2 and membrane-type 1 MMP
(MT1-MMP) are related to tumor invasion, metastasis, and
angiogenesis, and molecular imaging of their activity may be
helpful to predict the malignancy of tumors.3
Near-infrared (NIR) light, in the wavelength range of 650−
900 nm, is especially suitable for noninvasive in vivo imaging,4
because it is relatively poorly absorbed by biomolecules, so that
it can penetrate well through tissues, and background
autofluorescence is low in the NIR region.5 Several NIR
fluorescence probes for visualizing MMP activity in vivo have
been reported over the past decade. For example,
MMPsense680 was used to visualize MMP activity in tumor
tissue6 or atherosclerosis.7 However, all of these probes suffer
from various disadvantages. For example, most MMP probes
consist of a fluorophore containing sulfo groups, so the
activated probe after the enzymatic reaction with extracellular
RESULTS AND DISCUSSION
■
Design and Synthesis of NIR Fluorescence Probes for
MMP Activity. We employed Forster resonance energy
transfer (FRET) as a fluorescence-controlling off/on mecha-
nism. Specifically, we conjugated a fluorophore as a FRET
donor and a dark nonfluorescent quencher as a FRET acceptor
to the opposite terminals of a MMP peptide substrate. This
̈
Received: April 24, 2012
Published: July 25, 2012
© 2012 American Chemical Society
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Figure 1. (a) Design of our activatable MMP probe based on the FRET mechanism. (b) The strategy used to achieve long-term retention of the
MMP probe in the tissue after enzymatic reaction. The activated probe is cell permeable. Intracellular retention is dependent on the chemical
structure of the activated probe, i.e., the cell permeable fluorophore conjugated with the peptide residue.
Figure 2. (a) Chemical structures of MMP probes with the four NIR fluorophores. (b) Chemical structures of the expected MMP-generated
cleavage products of Cy5-MMP and BODIPY-MMP, i.e., Cy5-N and BODIPY-C, respectively.
FRET strategy is often used in fluorescence probes to detect
protease activity, because a very high signal-to-noise ratio can
easily be achieved, i.e., the fluorescence intensity of the
fluorophore is strongly suppressed before the enzymatic
reaction.12 In this study, we used BHQ-3 as the dark quencher,
together with four NIR fluorophores (Cy5, BODIPY650/665,
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Figure 3. (Top) Absorption spectra before (red) and after enzymatic reaction (blue) of the four MMP probes, Cy5-MMP (1 μM), SulfoCy5-MMP
(1 μM), SiR-MMP (1 μM), BODIPY-MMP (2 μM) in 1 mL TCN buffer containing 0.1% DMSO (for Cy5-MMP, SulfoCy5-MMP, and SiR-
MMP) or 3% DMSO (for BODIPY-MMP) as a cosolvent. (bottom) Fluorescence spectra of the MMP probes, Cy5-MMP (1 μM), SulfoCy5-MMP
(1 μM), SiR-MMP (1 μM), BODIPY-MMP (2 μM), incubated with 1 μg of MMP2 (catalytic domain recombinant protein) in 1 mL TCN buffer
containing 0.1% DMSO (for Cy5-MMP, SulfoCy5-MMP, and SiR-MMP) or 3% DMSO (for BODIPY-MMP) as a cosolvent at 37 °C for 300 min
(Cy5-MMP), 840 min (SulfoCy5-MMP), 600 min (SiR-MMP), or 240 min (BODIPY-MMP) with excitation at 620 nm (for BODIPY-MMP) or
640 nm (for Cy5-MMP, SulfoCy5-MMP, and SiR-MMP). Spectra were measured before (red) and after (blue) enzymatic reaction.
Figure 4. Bright field and fluorescence images of HT-1080 cells incubated with Cy5-MMP, SulfoCy5-MMP, SiR-MMP, or BODIPY-MMP (1 μM,
final 0.2% DMSO as a cosolvent) at 37 °C for 3 h in the presence (bottom) or the absence (top) of the inhibitor GM6001 (100 μM).
SiR650, and SulfoCy5) and a MMP substrate peptide as a
linker moiety (Figure 2a). Cy5 is one of the cyanine dyes,
which are typical NIR fluorophores.13 BODIPY650/665 is
based on a BODIPY (boron dipyrromethene) scaffold with a
relatively sharp NIR fluorescence peak and high quantum yield.
BODIPY dyes are relatively insensitive to the polarity and pH
of their environment.14 SiR650 is a novel NIR fluorophore
which is more resistant to bleaching than other NIR
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Figure 5. (a) Chemical structures of additionally synthesized compounds: peptide residues conjugated with various NIR fluorophores. (b) Flow
cytometric analysis of HT-1080 cells loaded with SulfoCy5-N (red), SiR-N (orange), Cy5-N (green), or BODIPY-C (blue). Each probe was used at
a final concentration of 1 μM in HBSS containing 0.1% DMSO as a cosolvent, and cells were incubated for 1 h at 37 °C. (c) Flow cytometric analysis
of HT-1080 cells incubated with BODIPY-N (green) or BODIPY-C (red) (final 1 μM, 0.1% DMSO as a cosolvent) at 37 °C. (d) Flow cytometric
analysis of HT-1080 cells incubated with BODIPY-C (final 1 μM, 0.1% DMSO as a cosolvent) for 1 h at 37 °C, washed with PBS, and then
incubated with HBSS for 0 min (green), 15 min (red), 30 min (blue), and 60 min (orange).
fluorophores.15 SulfoCy5, which has three sulfo groups, is also a
typical cyanine dye, and is cell impermeable due to its anionic
charges. Cy5, BODIPY650/665, and SiR650 are highly cell
permeable, and thus we anticipated that activated fluorescence
probes based on them would become cell permeable after
MMP enzymatic reaction. On the other hand, the MMP
fluorescence probe containing SulfoCy5 may be cell imperme-
able both before and after the MMP enzymatic reaction. We
synthesized SulfoCy5-MMP to assess the effect of the cell
permeability of the fluorophore on fluorescence imaging of
MMP activity in cultured live cells. The peptide linking the
fluorophore and dark quencher contains a PLGLAG peptide
sequence, which is selectively cleaved by MMP2, MMP9, and
MT1-MMP.16 Thus, we designed and synthesized four
candidate NIR fluorescence probes for MMP activity with the
four NIR fluorophores; the peptide linker was obtained by
Fmoc solid-phase peptide synthesis. We initially tried to
conjugate the fluorophore to the N-terminal and BHQ-3 to the
C-terminal of the peptide substrate, and successfully synthe-
sized Cy5-MMP, SulfoCy5-MMP, and SiR-MMP. However, it
proved difficult to synthesize the probe with BODIPY650/665
conjugated to the N-terminal of the peptide. So, only in the
case of BODIPY650/665, we designed and synthesized the
probe with the fluorophore at the C-terminal of the peptide
substrate (Figure 2b and Supporting Information [SI]).
was observed compared with the spectrum of the fluorophore
alone. This blue-shift of the absorption is considered to be due
to stacking interaction between the fluorophore and BHQ-3.
The fluorescence quantum yields of all the probes were less
0.001 owing to FRET and the stacking interaction.17 After
addition of MMP, the absorption spectrum of each probe
corresponded to the combined spectrum of the fluorophore
and BHQ-3, and the fluorescence intensities of all the MMP
probes also increased. The kinetic parameter of these probes
with MMP2, Vmax/Km, was determined by applying the
equation shown in Table S1 (SI). As compared with the
commercially available Omni fluorescence substrate, which is
often used to detect MMP activity in vitro, all the probes
seemed to be cleaved rapidly by MMP. HPLC analysis
confirmed that all the probes were cleaved by MMP2, as
expected (Figure S5−S8 [SI]).
Live Cell Fluorescence Imaging of MMP Activity. We
applied the four probes to cultured HT-1080 cells, which
express MMP2 and MT1-MMP,18 to assess their intracellular
retention after enzymatic reaction by means of fluorescence
microscopy. The cells were incubated with 1 μM MMP probe
for 180 min in the absence or presence of 100 μM GM6001,
which is a broad-spectrum MMP inhibitor. BODIPY-MMP
showed a large increase of intracellular fluorescence in the
absence of GM6001, and this fluorescence increment was
suppressed in the presence of GM6001 (Figure 4). On the
other hand, no significant difference of intracellular fluores-
cence was observed between the absence and presence of
GM6001 with the other probes, i.e., essentially no fluorescence
increment inside cells was observed, even though the
extracellular fluorescence intensity was increased (Figure 4).
From these results, it is considered that all the probes were
cleaved by MMPs, but Cy5-MMP, SulfoCy5-MMP, and SiR-
Fluorescence and Chemical Properties of the MMP
Probes. We examined the spectroscopic properties of the four
synthesized MMP probes under physiological conditions. The
absorption spectra of the probes changed after addition of
MMP2 or MT1-MMP in accordance with the change in
fluorescence emission spectra (Figure 3, and Figures S1−S4
[SI]), i.e., before addition of MMP, the fluorescence intensity of
each probe was negligible, and a blue-shift of the absorption
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MMP remained in the extracellular environment after the
enzymatic reaction. On the other hand, MMP-activated
BODIPY-MMP penetrated into the cells and afforded clear
cellular images. We further confirmed the high intracellular
retention of BODIPY-MMP by flow cytometric analysis (see
Figure S9, SI). It is difficult to completely rule out the
possibility that a part of the MMP probe enters the cells intact
and is cleaved by intracellular MMPs, such as MT1-MMP.
However, we think that most of the BODIPY-MMP molecules
were cleaved by extracellular MMP, as planned, because the
increase of fluorescence intensity inside the cultured cells was
suppressed by adding to the medium ethylenediaminetetra-
acetic acid (EDTA), which is cell impermeable and inhibits
only extracellular MMP activity (Figure S10, SI).19
Detailed Investigation of the Cell Permeability of
MMP Probes after Enzymatic Reaction with MMP. Next,
to further examine the cell permeability of BODIPY-MMP
after enzymatic reaction with MMP, we synthesized Cy5-N,
SulfoCy5-N, SiR-N, and BODIPY-C, corresponding to the
structures of the four probes after enzymatic reaction with
MMP, i.e., the fluorophores conjugated with the peptide
residue (Figure 5a). The fluorescence quantum yields of these
molecules were over 0.1 (Table 1). Cultured HT-1080 cells
lipophilicity and electric charge, while the effect of the peptide
residue may depend upon both the nature of the side chains
and the peptide terminal generated by MMP, i.e., carboxylic
acid or amino group.
We also examined the intracellular retention of BODIPY-C
in HT-1080 cells by means of flow cytometry. The intracellular
fluorescence of BODIPY-C was still intense even at 60 min
after excess probe had been washed out with phosphate-
buffered saline (PBS) (Figure 5d). The fluorescence intensity
of stained cells remained almost unchanged during incubation
for 0 to 60 min in PBS after washout of the extracellular
solution. We also examined the localization of BODIPY-C
inside cells, and found that fluorescence of BODIPY-C was
observed mainly in mitochondria and Golgi bodies (Figure S13,
SI). We think that this intracellular localization contributes to
the efficient long-term intracellular retention.
In Vivo Imaging in Tumor-Bearing Mouse. Finally, we
applied BODIPY-MMP to visualize the MMP activity of tumor
tissue in vivo. BODIPY-MMP was injected intratumorally into a
HT-1080 tumor-bearing mouse, and the change in the
fluorescence intensity was monitored (Figure 6). The
fluorescence of BODIPY-MMP was clearly detected at the
tumor after 15 min and was well maintained for over 6 h, while
the fluorescence of the SulfoCy5-MMP, of which the activated
form is cell impermeable, was detected at both the tumor and
throughout the whole body soon after the intratumoral
injection, presumably due to rapid diffusion of the dye over
the body, but the tumor was no longer detectable after 60 min.
To confirm that the activated probe after administration of
BODIPY-MMP was indeed present intracellularly in vivo, the
tumor at 360 min after intratumoral injection of BODIPY-
MMP was removed and imaged by confocal fluorescence
microscopy at the cellular level (Figure S14, SI). The
fluorescence of BODIPY-MMP was indeed detected inside
cells of the tumor tissue, as in the case of the experiments with
cultured HT-1080 cells. So, it is considered that BODIPY-
MMP was also cleaved by MMP in the tumor tissue and the
activated BODIPY-MMP, i.e., BODIPY-C, was introduced into
cells in vivo. We further assessed whether the increment of the
fluorescence intensity of BODIPY-MMP in vivo was due to
MMP enzymatic activity. To address this question, we newly
synthesized ControlBODIPY-MMP, which has the reverse
peptide sequence of the peptide substrate for MMP2, MMP9
and MT1-MMP, and is not cleaved by MMP.16 When we
intratumorally injected ControlBODIPY-MMP into the tumor,
only a minor fluorescence enhancement, which might be due to
the relative instability of BHQ-3,20 was observed compared
with the case of BODIPY-MMP injection (Figure 6).
Therefore, we consider that the difference of the fluorescence
enhancement of BODIPY-MMP and ControlBODIPY-MMP
reflected the MMP activity in vivo.
a
Table 1. Fluorescence Quantum Yields of the MMP Probes.
Cy5
SulfoCy5
SiR
BODIPY
MMP probes
<0.001
0.129
<0.001
0.179
<0.001
0.169
<0.001
0.127
synthetic cleaved MMP probe
a
Fluorescence quantum yields were determined in TCN buffer (pH
7.4), referring to that of cresyl violet (Φfl = 0.54) in methanol as a
standard.
were incubated with each probe (1 μM) for 60 min and
analyzed by flow cytometry. BODIPY-C gave the highest
intracellular fluorescence intensity among the four compounds
(Figure 5b). But, flow cytometric analysis is often influenced by
the inherent photophysical properties of fluorescent dyes, i.e.,
both the extinction coefficient and the fluorescence quantum
yield of the fluorophore. So, we next conducted fluorescence
imaging of HT-1080 cells incubated with the four synthesized
molecules by means of fluorescence microscopy with or
without a washout process (Figure S11, SI). Only BODIPY-
C clearly stained the cells both before and after washout of the
extracellular solution. On the other hand, Cy5-N, SiR-N, and
SulfoCy5-N poorly stained the cells, and the extracellular
solution showed intense fluorescence intensity. These results
indicate that BODIPY-C is highly cell permeable, whereas the
other compounds are insufficiently permeable to image the cells
clearly. These results are consistent with those of fluorescence
imaging with the MMP probes (Figure 4).
Next, to examine whether the long-term retention in the
tumor tissue was related to the cell permeability of BODIPY-C
or SulfoCy5-N, we intratumorally injected these compounds,
which are the products of the enzymatic reaction of BODIPY-
MMP or SulfoCy5-MMP with MMP, respectively, and the
time course of fluorescence intensity change at the tumor was
monitored (Figure 6c). Although the fluorescence of SulfoCy5-
N, which is cell impermeable, was quickly washed out from the
tumor tissue and disappeared from the whole body after 30
min, the fluorescence of BODIPY-C, which has high cell
permeability, was highly retained in the tumor even after 6 h
(data not shown). These results suggest that high cell
We further investigated the effect of the fluorophore-
conjugated terminal, i.e., the N-terminal or the C-terminal of
the peptide substrate, on the cell permeability of the activated
MMP probes. Compounds with N-terminal BODIPY650/665
(BODIPY-N) and C-terminal Cy5 (Cy5-C) (Figure S12, SI)
were further synthesized. Flow cytometric analysis indicated
that the C-terminal dye was more cell permeable than the N-
terminal dye for both BODIPY650/665 and Cy5 as a
fluorophore (Figures 5c and S12, SI). Thus, the cell
permeability of the MMP probe after enzymatic reaction
appears to depend on both the fluorophore and the conjugated
peptide. The effect of the fluorophore may be based on both its
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Figure 6. (a) Chemical structure of ControlBODIPY-MMP. (b) White light (left) and fluorescence (right) images of HT-1080 tumor-bearing nude
mouse at 60 min after the injection. BODIPY-MMP (top), SulfoCy5-MMP (middle) or ControlBODIPY-MMP (bottom) (10 μM in 20 μL PBS
containing 1% DMSO as a cosolvent) was administrated by intratumoral injection. Images were captured at 120 min after the injection. The site of
the tumor is indicated by red circles. (c) White light and fluorescence images of HT-1080 tumor-bearing nude mouse at 3 and 30 min after the
injection. BODIPY-C (top) or SulfoCy5-N (bottom) (1 μM in 20 μL PBS containing 0.1% DMSO as a cosolvent) was administered by
intratumoral injection.
permeability of the activated MMP probe after enzymatic
reaction is critical for long-term dye retention in the target
tissue and for in vivo fluorescence imaging with a high signal-to-
noise ratio.
design strategy will be applicable to a range of NIR fluorescence
probes, not only for MMP activity, but also other proteases.
EXPERIMENTAL SECTION
■
Reagents and solvents were of the best grade available, supplied by
Tokyo Chemical Industries, Wako Pure Chemical, Aldrich Chemical
Co., Biosearch Technology, Kanto Chemical Company, Watanabe
Chemical Industry, Applied Bioscience or Invitrogen, and were used
without further purification. Saline was purchased from Otsuka
Pharmaceutical Co. Ltd. Mice (BALB/cAJcl-nu/nu) were purchased
from CLEA Japan. Reactions were monitored by TLC with visual
observation of the dye spot or by HPLC. All compounds were purified
on a silica gel column and by HPLC. All fluorescent probes were
considered pure when a single HPLC peak was obtained.
CONCLUSIONS
■
In conclusion, we have designed, synthesized, and evaluated a
novel MMP probe, BODIPY-MMP. Our design approach was
to conjugate a fluorophore as a FRET donor and a dark
quencher as a FRET acceptor at the opposite ends of a MMP
peptide substrate. To achieve long-term retention of the probe
at the target site and a high signal-to noise ratio, we focused on
high cell permeability of the MMP-activated probe, which was
consequently expected to be accumulated intracellularly at the
target tissue following the extracellular MMP reaction, instead
of being washed out, as is the case for cell impermeable
activated probes. BODIPY-MMP could clearly visualize MMP
activity as a fluorescence increment inside cultured HT-1080
cells. Further, following intratumoral injection of BODIPY-
MMP in a mouse xenograft tumor model, the MMP activity
could be monitored (i.e., the tumor could be visualized) for
much longer than the case with the probe containing cell
impermeable SulfoCy5. In contrast to previously reported
probes, this probe offers the advantages of rapid and sustained
response with a high signal-to-noise ratio. We think this simple
1
Instruments. H or 13C NMR spectra were recorded on a JEOL
JNM-LA300 or JMN-LA400. Mass spectra were measured with a
JEOL JMS-T1000LC mass spectrometer (ESI+ and ESI−). HPLC
purification and analyses were performed on reversed-phase columns
(GL Science (Tokyo, Japan), Inertsil ODS-3 10 mm ×250 mm for
purification and Inertsil ODS-3 4.6 mm ×250 mm for analyses) fitted
on a Jasco PU-2080 system and eluted with eluent A (H2O containing
0.1% TFA (v/v)) and eluent B (CH3CN with 20% H2O containing
0.1% TFA (v/v)). Details of HPLC conditions are included with the
synthesis and characterization data. Absorption spectra were obtained
with a Shimadzu UV-1650 (Tokyo, Japan). Fluorescence spectroscopic
studies were performed with a Hitachi F4500 (Tokyo, Japan). The slit
widths were 2.5 nm for excitation and 5 nm for emission. The
photomultiplier voltage was 700 V. Peptide synthesis was performed
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with 433A peptide Synthesizer (Applied Biosystems, U.S.A.) and
SynthAssist 3.1 (Applied Biosystems, U.S.A.).
images were captured without washout of excess probe at 15 min after
addition of the probe to the medium.
Photochemical Properties and Fluorescence Quantum
Yield. Photochemical properties of dyes were examined in TCN
buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl) (pH 7.5)
containing 0.1% DMSO as a cosolvent, using a Shimadzu UV-1650
UV−vis spectrometer and a Hitachi F4500 fluorescence spectrometer.
For determination of the fluorescence quantum yield (Φfl), cresyl
violet in methanol (Φfl = 0.54) was used as a standard, and the results
were calculated according to the following equation (subscript “st”
stands for the reference, and “x”, for the sample).
For fluorescence microscopic imaging with the synthesized cleaved
probes, HT-1080 cells seeded on an 8-chamber plate (Thermo
Scientific, U.S.A.) were washed with PBS, then 200 μL HBSS was
added. After that, Cy5-N, SulfoCy5-N, SiR-N, or BODIPY-C (final 1
μM, DMSO 0.1% as a cosolvent) was added to the medium, and
incubation was continued at 37 °C. Fluorescence images were
captured without washout of excess probe at 1 h after addition of the
probe to the medium. The HT-1080 cells were washed with PBS, then
200 μL HBSS was added, and fluorescence images were captured with
an Olympus IX 71 equipped with a cooled CCD camera (Coolsnap
HQ, Olympus) and a xenon lamp (AH2RX-T, Olympus) with a Cy5
excitation filter and a Cy5 emission filter.
Φ /Φ = [Ast/Ax][nx2/ns2t][D /D ]
x
st
x
st
Flow Cytometric Analysis. HT-1080 cells plated on a multiwell
12-well plate (Falcon) were washed with PBS, then 1 mL HBSS was
added. GM6001 (final 100 μM containing 0.1% DMSO as a
cosolvent) was added to the medium in the case of the sample with
the inhibitor, whereas 1 μL DMSO was added to the medium in the
case of the sample without the inhibitor. The samples were incubated
at 37 °C for 15 min. Then, the sample was incubated with synthesized
probe (final 1 μM, 0.2% DMSO as a cosolvent) in HBSS at 37 °C for 1
h. The cells were washed with PBS and trypsinized. HBSS was added,
and the fluorescence intensity was measured with a BD LSR 2 flow
cytometer (BD Biosciences) and FlowJo (Digital Biology). The
excitation wavelength was 633 nm, and the emission was detected with
an APC filter 660 20 nm.
For examination of the cell permeability of the synthesized cleaved
MMP probes, HT-1080 cells on a multiwell 12-well plate (Falcon)
were washed with PBS, then 1 mL HBSS was added. Then, the sample
was incubated with synthesized cleaved probe (final 1 μM, 0.1%
DMSO as a cosolvent) in HBSS at 37 °C for 1 h. The cells were
washed with PBS and trypsinized. HBSS was added, and the
fluorescence intensity was measured with a BD LSR 2 flow cytometer
(BD Biosciences) and FlowJo (Digital Biology).
Analyses of Intracellular Retention of BODIPY-C. HT-1080
cells plated on a multiwell 12-well plate (Falcon) were washed with
PBS, then 1 mL HBSS was added. The sample was incubated with
BODIPY-C (final 1 μM, 0.1% DMSO as a cosolvent) in HBSS at 37
°C for 1 h. Then, the cells were washed with PBS and trypsinized.
HBSS was added, and flow cytometric analysis was performed with a
BD LSR 2 flow cytometer (BD Biosciences) and FlowJo (Digital
Biology). The time course of fluorescence intensity change was
measured at 0, 15, 30, and 60 min after addition of HBSS.
Fluorescence Confocal Microscopy. HT-1080 cells seeded on
an 8-chamber plate (Thermo Scientific, U.S.) were washed with PBS,
then incubated in HBSS containing 0.5 μM NBD C6-ceramide-BSA at
4 °C for 60 min. The cells were washed with PBS, and incubated in
HBSS containing 1 μM BODIPY-C and 0.5 μM MitoTracker red
containing 0.2% (v/v) DMSO as a cosolvent at 37 °C for 30 min.
Fluorescence images were captured using a Leica TCS SP5 confocal
microscope equipped with a 40× objective lens using Leica
Application Suite Advanced Fluorescence (LAS-AF) software. The
light source was a white-light laser. The excitation wavelength was 470
nm (NBDC6-ceramide-BSA), 579 nm (MitoTracker red), and 640 nm
(BODIPY-C), and emission wavelength was 492−542 nm (NBDC6-
ceramide-BSA), 592−620 nm (MitoTracker red), and 659−751 nm
(BODIPY-C). PMT; Gain: 1065 V (NBDC6-ceramide-BSA), 1000 V
(MitoTracker red) and 802 V (BODIPY-C), Offset 0%.
where A = absorbance at the excitation wavelength; n = refractive
index; and D = area under the fluorescence spectra on an energy scale.
Enzyme Assay. One microgram MMP2 catalytic domain (human)
(ENZO Life Science, U.S.A.) in 2 μL TCNB buffer (50 mM Tris, 10
mM CaCl2, 150 mM NaCl, 0.05% Brij 35) (pH 7.5) or 1 μg MT1-
MMP catalytic domain (human) (Calbiochem, U.S.A.) in 2 μL TCNB
buffer was incubated for 15 min at 37 °C before the enzyme assay.
Each probe was used at a final concentration of 1 or 2 μM in 1 mL
TCN buffer containing 0.1% (Cy5-MMP, SulfoCy5-MMP, SiR-
MMP) or 3% (BODIPY-MMP) DMSO as a cosolvent at 37 °C.
Fluorescence spectra were measured every 10 min after the enzyme
solution was added until termination of the enzymatic reaction.
HPLC Analysis. Ten micromolar synthesized MMP probe was
dissolved in 1 mL TCN buffer containing 1% DMSO as a cosolvent,
and 1 μg MMP2 catalytic domain (ENZO Life Science) was added to
the solution, which was then incubated at 37 °C for 6 h. The solution
was lyophilized, and 30 μL DMF/30 μL TCN buffer was added to the
residue. This solution was analyzed by HPLC. HPLC analysis: eluent,
a 20- or 40-min linear gradient from 20% to 100% solvent B; flow rate,
1.0 mL/min; detection wavelength, 640 nm.
Determination of Kinetic Parameters. Synthesized MMP probe
(0.1 μM) was dissolved in 3 mL TCN buffer containing 0.1% DMSO
as a cosolvent, and the solution was incubated at 37 °C. One
microgram MMP2 (human) (Sigma, U.S.A.) in 20 μL TCNB buffer
incubated at 37 °C for 15 min was added to the solution, and the
fluorescence intensity change was recorded at 37 °C. The time course
of fluorescence intensity was fitted to the equation shown in Table S1
in the Supporting Information to determine Vmax/Km.
Cell Lines and Culture Conditions. Human fibrosarcoma cell
line HT-1080 was purchased from the Health Science Research
Resource Bank (Osaka, Japan). HT-1080 cells were cultured in EMEM
(Eagle’s minimal essential medium) (Wako), containing nonessential
amino acids (Invitrogen), 10% fetal bovine albumin (Invitrogen) and
1% penicillin/streptomycin (Invitrogen). Cultured cells were
incubated at 37 °C in an atmosphere of 5% CO2 in air.
Fluorescence Microscopic Imaging with MMP Probes. HT-
1080 cells seeded on an 8-chamber plate (Thermo Scientific, U.S.A.)
were washed with PBS, then 200 μL HBSS was added. GM6001 (final
100 μM containing 0.1% DMSO as a cosolvent) was added to the
medium in the case of the sample with the inhibitor, while 0.2 μL
DMSO was added to the medium in the case of the sample without
the inhibitor. The samples were incubated at 37 °C for 15 min. Then,
Cy5-MMP, SulfoCy5-MMP, SiR-MMP, or BODIPY-MMP (final 1
μM, DMSO 0.2% as a cosolvent) was added to the medium, and
incubation was continued at 37 °C. Fluorescence images were
captured without washout of excess probe at 3 h after addition of the
probe to the medium.
Mouse Models. All procedures were approved by the Animal Care
and Use Committee of the University of Tokyo. Fluorescence imaging
with synthesized MMP probes was performed in BALB/cAJcl-nu/nu
mice (6−7 weeks, ♀), each bearing a HT-1080 xenograft tumor
prepared by injection of two million cells into the left hind leg at 7−14
days before the probe injection.
In Vivo Fluorescence Imaging of Mice after Intratumoral
Injection of Fluorescence Probes. All procedures were approved
by the Animal Care and Use Committee of the University of Tokyo.
After intratumoral injection of 10 μM BODIPY-MMP, SulfoCy5-
MMP, or ControlBODIPY-MMP in 20 μL PBS containing 1%
For fluorescence microscopic imaging with BODIPY-MMP and
EDTA, HT-1080 cells seeded on an 8-chamber plate (Thermo
Scientific, U.S.A.) were washed with PBS, then 200 μL HBSS was
added. 0.1 M EDTA2Na in 2 μL PBS (final 1 mM) was added to the
medium in the case of the sample with the inhibitor, whereas 2 μL PBS
was added to the medium in the case of the sample without the
inhibitor. The samples were incubated at 37 °C for 15 min. Then,
BODIPY-MMP (final 1 μM, DMSO 0.1% as a cosolvent) was added
to the medium, and incubation was continued at 37 °C. Fluorescence
13736
dx.doi.org/10.1021/ja303931b | J. Am. Chem. Soc. 2012, 134, 13730−13737

Journal of the American Chemical Society
Article
DMSO as a cosolvent, bright field and fluorescence images were
captured at different time points with a Maestro In-vivo Imaging
System (CRi Inc., Woburn, MA), with an excitation filter of 635 nm,
an emission filter of 675 nm, and bright field settings. All image
analyses were performed using Image J (NIH, Bethesda, MD, U.S.A.).
For in vivo imaging of mice with the synthesized cleaved MMP
probe, 1 μM BODIPY-C or SulfoCy5-N in 20 μL PBS containing
0.1% DMSO as a cosolvent, was administered by intratumoral
injection, and bright field and fluorescence images were captured at
different time points with the same instruments.
Confocal Fluorescence Imaging of Live Mouse Tumor
Tissue. All procedures were approved by the Animal Care and Use
Committee of the University of Tokyo. Mice were sacrificed and the
tumors removed and kept on ice in HBSS until imaging. Each tumor
was sliced, placed on a 35 mm glass-bottomed dish, and incubated
with Calcein-AM (final 1 μM, 0.1% DMSO as a cosolvent) in HBSS
for 30 min, then imaged by confocal microscopy. The excitation
wavelengths were 493 nm (Calcein) and 640 nm (BODIPY-MMP),
and emission wavelengths were 502−562 nm (Calcein) and 659−751
nm (BODIPY-MMP), respectively. PMT; Gain: 800 V (Calcein), 550
V (BODIPY-MMP), Offset 0%.
REFERENCES
■
(1) (a) Seiki, M. Curr. Opin. Cell. Biol. 2002, 14, 624−632. (b) Seiki,
M.; Itoh, Y. J. Cell. Phys. 2006, 206, 1−8. (c) Seiki, M. Cancer Lett.
2003, 194, 1−11.
(2) (a) Chakraborti, S.; Mandal, M.; Das, S.; Mandal, A.;
Chakraborti, T. Mol. Cell. Biol. 2003, 253, 269−285. (b) Shiomi, T.;
Lemaïtre, V.; D’Armiento, J.; Okada, Y. Pathol. Int. 2010, 60, 477−496.
(c) Visse, R.; Nagase, H. Circ. Res. 2003, 92, 827−839. (d) Werb, Z.;
Egeblad, M. Nat. Rev. 2002, 2, 161−174.
(3) Gialeli, C.; Theocharis, A. D.; Karamanos, N. K. FEBS J. 2011,
278, 16−27.
(4) Scherer, R. L.; McIntyre, J. O.; Matrisian, L. M. Can. Met. Rev.
2008, 27, 679−690.
(5) Weissleder, R. Nat. Biotechnol. 2001, 19, 316−317.
(6) Bremer, C.; Tung, C. H.; Weissleder, R. Nat. Med. 2001, 7, 743−
748.
(7) Deguchi, J.; Aikawa, M.; Tung, C.; Aikawa, E.; Kim, D.;
Ntziachristos, V.; Weissleder, R.; Libby, P. Circulation 2006, 114, 55−
62.
(8) Yamane, T.; Hanaoka, K.; Muramatsu, Y.; Tamura, K.; Adachi, Y.;
Miyashita, Y.; Hirata, Y.; Nagano, T. Bioconjugate Chem. 2011, 22,
2227−2236.
(9) (a) Aguilera, T. A.; Olson, E. S.; Timmers, M. M.; Jiang, T.;
Tsien, R. T. Integr. Biol. 2009, 1, 371−381. (b) Olson, E. S.; Aguilera,
R. A.; Jiang, T.; Ellies, L. G.; Nguyen, Q. T.; Wong, E. H.; Gross, L. A.;
Tsien, R. Y. Integr. Biol. 2009, 1, 382−393.
(10) Jones, S. W.; Christison, R.; Bundell, K.; Voyce, C. J.;
Brockbank, S. M. V.; Newham, P.; Lindsay, M. A. Br. J. Pharmacol.
2005, 145, 1093−1102.
(11) Kaplan, I. M.; Wadia, J. S.; Dowdy, S. F. J. Controlled Release
2005, 102, 247−253.
(12) Razkin, J.; Josserand, V.; Boturyn, D.; Jin, Z.; Dumy, P.; Favrot,
M.; Coll, J.; Texier, I. ChemMedChem 2006, 1, 1069−1072.
(13) (a) Tung, C. H.; Lin, Y.; Moon, W. K.; Weissleder, R.
ChemBioChem 2002, 8, 784−786. (b) Becker, A.; Hessenius, C.; Licha,
K.; Ebert, B.; Sukowski, U.; Semmler, W.; Wiedenmann, B.;
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed descriptions of synthetic procedures for the MMP
probes; data on the measurements of absorption and
fluorescence spectra of the MMP probes treated with MT1-
MMP; HPLC chromatograms of the MMP probes, the reaction
mixture of the MMP probes incubated with MMP2, and the
synthesized cleaved MMP probes; Vmax/Km values of the MMP
probes for MMP2; flow cytometric analysis of HT-1080 cells
loaded with the MMP probes in the presence or the absence of
MMP inhibitor; DIC and fluorescence images of HT-1080 cells
stained with BODIPY-MMP in the presence or absence of
EDTA; fluorescence images of HT-1080 cells incubated with
the MMP probe residue; fluorescence images of HT-1080 cells
incubated with BODIPY-C, NBDC6-ceramide-BSA, and
MitoTracker red; bright field and confocal fluorescence images
of live mouse tumor tissue loaded with Calcein-AM and
BODIPY-MMP; white light and fluorescence images of HT-
1080 tumor-bearing mouse loaded with BODIPY-C or
SulfoCy5-N. This material is available free of charge via the
Internet at http://pubs.acs.org.
Grotzinger, C. Nat. Biotechnol. 2001, 19, 327−331. (c) Zheng, G.;
̈
Li, H.; Yang, K.; Blessington, D.; Licha, K.; Lund-Katz, S.; Chance, B.;
Glickson, J. D. Bioorg. Med. Chem. Lett. 2002, 12, 1485−1488.
(d) Chen, X. Y.; Conti, P. S.; Moats, R. A. Cancer Res. 2004, 64, 8009−
8014. (e) Ke, S.; Wen, X. X.; Gurfinkel, M.; Charansangavej, C.;
Wallace, S.; Sevick-Muraca, E. M.; Li, C. Cancer Res. 2003, 63, 7870−
7875. (f) Ozmen, B.; Akkaya, E. U. Tetrahedron Lett. 2000, 41, 9185−
9188. (g) Sasaki, E.; Kojima, H.; Nishimatsu, H.; Urano, Y.; Kikuchi,
K.; Hirata, Y.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 3684−3685.
(h) Weissleder, R.; Tung, C.; Mahmood, U.; Bogdanov, A., Jr. Nat.
Biotechnol. 1999, 17, 375−378. (i) Tung, C. H.; Bredow, S.;
Mahmood, U.; Weissleder, R. Bioconjugate Chem. 1999, 10, 892−896.
(14) Loudet, A.; Burgess, K. Chem. Rev. 2007, 107, 4891−4932.
(15) (a) Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T.
ACS Chem. Biol. 2011, 6, 600−608. (b) Fu, M.; Xiao, Y.; Qian, X.;
Zhao, D.; Xu, Y. Chem. Commun. 2008, 15, 1780−1782.
AUTHOR INFORMATION
■
Corresponding Author
tlong@mol.f.u-tokyo.ac.jp
Notes
(16) Jiang, T.; Olson, E. S.; Nguyen, Q. T.; Roy, M.; Jennings, P. A.;
Tsien, R. Y. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17867−17872.
(17) Johansson, M. K.; Cook, R. M. Chem. Eur. J. 2003, 9, 3466−
3471.
(18) Zhao, T.; Harada, H.; Teramura, Y.; Tanaka, S.; Itasaka, S.;
Morinibu, A.; Shinomiya, K.; Zhu, Y.; Hanaoka, H.; Iwata, H.; Saji, H.;
Hiraoka, M. J. Controlled Release 2010, 144, 109−114.
(19) (a) Komatsu, K.; Kikuchi, K.; Kojima, H.; Urano, Y.; Nagano, T.
J. Am. Chem. Soc. 2005, 127, 10197−10204. (b) Cheng, D.; Shen, Q.;
Nan, F.; Qian, Z.; Ye, Q. Protein Expr. Purif. 2003, 27, 63−74.
(20) Linder, K. E.; Metcalfe, E.; Nanjappan, P.; Arunachalam, T.;
Ramos, K.; Skedzielewski, T. M.; Marinelli, E. R.; Tweedle, M. F.;
Nunn, A. D.; Swenson, R. E. Bioconjugate Chem. 2011, 22, 1287−1297.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported in part by the Ministry of
Education, Culture, Sports, Science and Technology of Japan
(Specially Promoted Research Grant Nos. 22000006 to T.N.
and 20689001, 19890047, 21659024, and 24659042 to K.H.),
and SENTAN, JST (to K.H). K.H. was also supported by
Grant-in-Aid from the Tokyo Biochemical Research Founda-
tion, Inoue Foundation for Science, Takeda Science Founda-
tion, the Research Foundation for Pharmaceutical Sciences,
Konica Minolta Science and Technology Foundation, The
Asahi Glass Foundation and Astellas Foundation for Research
on Metabolic Disorders.
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dx.doi.org/10.1021/ja303931b | J. Am. Chem. Soc. 2012, 134, 13730−13737