A two-photon tracer for glucose uptake

Article abstract of DOI:10.1002/anie.200901175

It takes two: Two-photon tracers (AG1, AG2) have been developed that can be excited by 780 nm fs laser pulses, can be easily loaded into cancer cells and tissues, show high photostability and negligible toxicity, and can visualize glucose uptake in living

Full text of DOI:10.1002/anie.200901175

Angewandte
Chemie
DOI: 10.1002/anie.200901175
Fluorescent Probes
A Two-Photon Tracer for Glucose Uptake**
Yu Shun Tian, Hyang Yeon Lee, Chang Su Lim, Jongmin Park, Hwan Myung Kim, Yoo Na Shin,
Eun Sun Kim, Hoon Jae Jeon, Seung Bum Park,* and Bong Rae Cho*
Glucose is the principal energy source essential for cell
growth. Fast-growing cancer cells exhibit a high rate of
glycolysis; hence, the rate of glucose uptake is faster in
these cells, primarily due to overexpression or
enhanced intracellular translocation of glucose trans-
porters (GLUTs) and increased activity of mitochon-
dria-bound hexokinases in the tumor.[1] To monitor
glucose metabolism in living systems, a variety of
tracers, such as [18F]-2-fluoro-2-deoxyglucose (18FDG),
2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-2-
deoxy-d-glucose (2-NBDG; Scheme 1), and IR dye
800CW-2DG, have been developed. 18FDG is widely
used in the in vivo analysis of glucose metabolism by
positron emission tomography (PET),[2–4] whereas
2-NBDG and IR dye 800CW-2DG are fluorescent
probes that have been used for studying cellular
metabolic functions involving GLUTs and in tumor-
imaging studies.[5–8] Recently, we developed a new
fluorescent probe, cyanine-3-linked O-1-glycosylated
glucose (Cy3-Glc-a; Scheme 1), which is a better glucose
probe than 2-NBDG because it can be used without glucose
starvation, produces a much brighter image, and can be
applied for the screening of anticancer agents.[9]
In one-photon microscopy (OPM), the probes are excited
with short-wavelength light ( ꢀ 350–550 nm); this, however,
limits their application in tissue imaging, owing to inherent
problems such as shallow penetration depth (< 80 mm),
Scheme 1. Structures of fluorescent tracers AG1, AG2, 2-NBDG, and Cy3-Gly-a.
interference by cellular autofluorescence, photobleaching,
and photodamage.[10,11] To overcome these problems, it is
crucial to use two-photon microscopy (TPM), which utilizes
two near-infrared photons for excitation. TPM offers a
number of advantages over OPM, including greater penetra-
tion depth (> 500 mm), localized excitation, and longer
observation times.[12,13] In particular, the extra penetration
depth afforded by TPM is an essential element for application
in tissue-imaging studies because the artifacts arising from
surface preparation, such as damaged cells, can extend over
70 mm into the tissue interior.[14] However, visualization of
glucose uptake by live cells and tissues with two-photon (TP)
tracers has not been reported so far.
[*] H. Y. Lee, J. Park, Prof. Dr. S. B. Park
Department of Chemistry, Seoul National University
Seoul 151-747 (Korea)
Fax: (+82)2-884-4025
E-mail: sbpark@snu.ac.kr
The requirements for a TP tracer to visualize glucose
uptake include sufficient water solubility for staining cells and
tissues, preferential uptake by cancer cells, a large TP cross-
section for a bright TPM image, pH resistance, and high
photostability. Our strategy was to link a-d-glucose with the
fluorophore 2-acetyl-6-dimethylaminonaphthalene (acedan)
through 3,6-dioxaoctane-1,8-diamine or a piperazine linkage
(in AG1 and AG2, respectively; Scheme 1), so that the tracers
are transported into the cells through the glucose-specific
mechanism. Acedan is a polarity-sensitive fluorophore that
has been successfully employed in the development of TP
probes for the cell membrane,[15] metal ions,[16–18] and acidic
vesicles.[19] We now report that these tracers facilitate the
visualization of glucose uptake in cancer cells and live tissues
at a depth of 75–150 mm for more than 3000 s and can be used
for screening anticancer agents.
Dr. Y. S. Tian, C. S. Lim, Y. N. Shin, Prof. Dr. B. R. Cho
Department of Chemistry, Korea University
1-Anamdong, Seoul 136-701 (Korea)
Fax: (+82)2-3290-3544
E-mail: chobr@korea.ac.kr
Prof. Dr. H. M. Kim
Department of Chemistry, Ajou University (Korea)
Dr. E. S. Kim, Prof. Dr. H. J. Jeon
School of Medicine, Korea University
[**] This work was partly supported by a Korea Science and Engineering
Foundation (KOSEF) grant funded by the Korea Ministry of
Education, Science, and Technology (MESF) (grant no.: R0A-2007-
000-20027-0), the WCU program through the KOSEF funded by the
MESF, and the MarineBio Technology Program funded by the
Ministry of Land, Transport, and Maritime Affairs (MLTM), Korea.
H.Y.L. and J.P. received BK21 Scholarships and Seoul Science
Fellowships.
The preparation of AG1 and AG2 is shown in Scheme 2.
6-Acetyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200901175.
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Scheme 2. a) N-Boc-3,6-dioxaoctane-1,8-diamine, Et3N, DMF, 508C;
b) N-Boc-piperazine, Et3N, DMF, 508C; c) 1. NaOMe, MeOH; 2. 50%
TFA/CH2Cl2; 3. 1, EDC, DIPEA, DMF. Boc: tert-butoxycarbonyl; DMF:
N,N-dimethylformamide; TFA: trifluoracetic acid; EDC: 3-(3-dimethyl-
aminopropyl)-1-ethylcarbodiimide; DIPEA: N,N-diisopropylethylamine.
(1), 2, and 3 were prepared by the method used in our
previous studies.[4,15–19] The reaction of 2 with N-Boc-piper-
azine afforded 4 in 70% yield. AG1 and AG2 were prepared
in 26 and 86% yields, respectively, by treating 3 and 4 with 1
(see the Supporting Information).
The fluorescence spectra of AG1 and AG2 showed
gradual bathochromic shifts with increases in the solvent
polarity (ENT) with the following order of solvents: 1,4-
dioxane < DMF < EtOH < H2O (Figure S1 and Table S1 in
the Supporting Information). The large bathochromic shifts
upon increases in the solvent polarity indicate the utility of
these molecules as polarity probes. Both compounds show
strong fluorescence in all of the solvents. Moreover, they are
pH insensitive in the biologically relevant pH range (Fig-
ure S1c and S1 f in the Supporting Information). The TP
action spectrum of AG1 determined by the two-photon-
excited fluorescence (TPEF) method[20] indicated a Fd value
of ꢀ 90 GM at 780 nm, which is much larger than those
observed for Cy3-Glc-a and 2-NBDG (Figure 1 and Table 1).
Hence, the TPM images of the samples stained with AG1 or
AG2 would appear much brighter than those stained with
Cy3-Glc-a or 2-NBDG.
The optimum concentration of these probes for the
cellular uptake experiments was determined by comparing
the TPM images of A549 cells treated with 6, 12.5, 25, 50, and
100 mm of AG1 and AG2 for 30 min. The TPM images
appeared brighter as the probe concentration was increased
up to 50 mm; however, at 100 mm concentrations, some cell
death was observed (Figure S2 in the Supporting Informa-
tion). Moreover, a higher uptake rate and a brighter TPM
image were obtained when the cells were treated with AG2
than when they were treated with AG1 (see Figure 3A and
Figure S3 in the Supporting Information). Furthermore, the
effect of AG2 on the viability of cells was studied by using the
CCK-8 kit: AG2 showed negligible toxicity, which indicates
that it could be applied for live-cell imaging (Figure S4 in the
Supporting Information). Therefore, we used 50 mm AG2 as
the optimum concentration in further cellular uptake experi-
ments.
Figure 1. A) One-photon absorption and emission spectra of AG1 and
AG2 in phosphate-buffered saline (PBS) buffer (137 mm NaCl, 3 mm
KCl, 10 mm Na2HPO4, 2 mm KH2PO4, pH 7.4). B) Two-photon action
spectra for AG1, AG2, Cy3-Glc-a, and 2-NBDG in PBS buffer.
Table 1: Photophysical data for AG1, AG2, Cy3-Glc-a, and 2-NBDG.[a]
ð1Þ
ðflÞ [b]
ð2Þ [d]
[e]
[f]
Compound
l
=l
F[c]
l
dmax
Fdmax
max
max
max
AG1
AG2
Cy3-Glc-a
2-NBDG
373/501
375/501
545/555
465/540[i]
0.90
0.56
0.01
n.d.[g]
780
95
86
88
780
155
n.d.[g]
n.d.[g]
n.d.[g,h]
n.d.[g,h]
n.d.[g,h]
n.d.[g,h]
[a] All measurements were performed in PBS buffer. [b] lmax values of the
one-photon absorption and emission spectra in nm. [c] Fluorescence
quantum yield, ꢁ15%. [d] lmax value of the two-photon absorption
spectrum in nm. [e] The peak two-photon action cross-section in
10ꢂ50 cm4/photon (GM), ꢁ15%. [f] Two-photon action cross-section in
GM. [g] n.d.: not determined. [h] The two-photon-excited fluorescence
intensity was too weak to allow accurate measurement of the cross-
section. [i] Reference [25].
red), respectively. The TPEF spectrum of the intense spots
was asymmetrical and could be fitted to two Gaussian
functions with emission maxima at 445 (green line in
Figure 2B) and 488 nm (orange line), whereas the TPEF
spectrum of the homogeneous domain could be fitted to a
single Gaussian function (pink line) with an emission
maximum at 497 nm. The longer wavelength band of the
dissected Gaussian function (orange line in Figure 2B) is
similar to the band of the single Gaussian function (pink line).
This result suggests that the probe is located in two regions of
different polarity: a more polar one that is likely to be cytosol
and a less polar one that is likely to be membrane associated.
Moreover, the shorter wavelength band (green in Figure 2B)
in the dissected Gaussian functions decreases to the baseline
at wavelengths of less than 520 nm. Consistently, the TPM
image collected at 520–620 nm is homogeneous without
The pseudocolored TPM images of cultured A549 cells
treated with 50 mm AG2 showed intense spots and homoge-
neous domains with two-photon emission maxima at 461
(marked in blue in Figure 2A and B) and 497 nm (marked in
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Figure 3. A) Relative AG1 and AG2 uptake by A549 cells. B) Relative
AG2 uptake in cancer cells (A549, HeLa) and normal cells (HEK293,
NIH/3T3). C) Relative AG2 uptake by A549 cells after treatment with
taxol (9.8 mm) for 0, 3, 6, 12, and 24 h. Cells were incubated with AG1
or AG2 (50 mm) for 10 min, either before (A and B) or after (C) treating
the cells with taxol (9.8 mm) for the designated period of time, and
TPEF intensities from 40–60 cells were determined by photomultiplier
tube. The data are the average of at least five independent experi-
ments.
Figure 2. Pseudocolored TPM images of A549 cells incubated with
50 mm AG2 for 10 min, collected at A) 360–620, C) 360–460, and
D) 520–620 nm. B) Two-photon-excited fluorescence spectra from the
hydrophobic (marked in blue) and hydrophilic (marked in red) regions
of the AG2-labeled A549 cells. The thin pale blue and pink curves
represent the dissected Gaussian functions for the blue and red
bands, respectively. The orange and green lines are discussed in the
text. The excitation wavelength was 780 nm. The images shown are
representative of the images obtained in the repeat experiments
(n=5). Scale bar: 30 mm.
uptake was not influenced by the presence of 55 mm d,l-
alanine, which indicates that osmotic pressure does not have
an impact on AG2 uptake. These results suggest that AG2 is a
glucose analogue that is taken up by the cells through a
glucose-specific transport system and not by passive diffu-
sion.[1,9,22,23] Therefore, AG2 can be used for visualizing
glucose uptake in living cells by TPM, without subjecting
the cells to glucose starvation.
To demonstrate the utility of this probe, we investigated
the effects of taxol on AG2 uptake by A549 cells. It was
expected that the anticancer agent would depress the cellular
metabolism and thereby reduce glucose uptake in the cancer
cells.[9] The cells were incubated with the anticancer agent for
1, 3, 6, 12, and 24 h, washed with PBS, treated with AG2, and
then imaged. The cellular uptake of AG2 was lower with
longer incubation times (Figure 3C and Figure S7B in the
Supporting Information). Dose-dependent uptake of AG2
was also observed with taxol concentrations of 49 nm, 490 nm,
and 9.8 mm (Table 2). Another anticancer agent, combretas-
tatin, also inhibited AG2 uptake in a similar manner
(Table 2). Hence, AG2 can be used for screening anticancer
agents in live cells by TPM.
intense spots (Figure 2D), whereas the one collected at 360–
460 nm clearly shows them (Figure 2C). Similar results were
reported for acedan-derived TP probes for Mg2+ (AMg1)[16]
and Ca2+ ions (ACa1).[17] Therefore, cytosolic AG2 can be
selectively detected by using the detection window of 520–
620 nm, with minimal interference from the membrane-
bound probes. Furthermore, the TPM images of A549 cells
costained with AG2 and MitoTracker, a well-known one-
photon fluorescent (OPF) probe for mitochondria,[21] merged
well with the OPM image (Figure S5 in the Supporting
Information), which indicates that the probes are predom-
inantly located in the mitochondria.
To assess whether AG2 is selectively taken up by cancer
cells, the efficiencies of AG2 uptake by A549 (lung carcinoma
cell line), HeLa (cervical cancer cell line), HEK293 (human
embryonic kidney 293 cell line), and NIH/3T3 (murine
fibroblast cell line) cells were compared. AG2 uptake was
most efficient in A549 and HeLa cells, followed by HEK293
and NIH/3T3 cells (Figure 3B and Figure S7A in the Sup-
porting Information); this result is similar to that obtained in
the case of Cy3-Glc-a[9] and confirms the selective uptake of
AG2 by cancer cells with enhanced glucose metabolism. The
cellular uptake experiment showed that AG2 effectively
competes with d-glucose in the media for cellular uptake; the
fluorescence intensities of AG2 in A549 cells inoculated with
media containing 10 mm or 50 mm d-glucose were reduced by
about 16 and 74%, respectively, as compared to that with
glucose-depleted medium, and the fluorescence intensity was
not affected by medium containing l-glucose (50 mm; Fig-
ure S6 in the Supporting Information). Furthermore, AG2
Table 2: Dose dependence of AG2 uptake by A549 cells in the presence
of various concentrations of anticancer agents.
AG2 uptake [%]
after 6 h
after 12 h
taxol (9.8 mm)
taxol (490 nm)
taxol (49 nm)
combretastatin (2 mm)
69.5
89.6
97.6
70.2
50.7
69.5
81.8
53.2
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We further assessed the utility of AG2 in tissue imaging
tissue surface. The TPEF intensities appear scattered at
shallow depths due to different degrees of dye adsorption at
tissue surfaces that are very different, as shown in the bright-
field images (Figure 4A and Figure S8A in the Supporting
Information). However, we were able to obtain reliable
results at a depth of ꢃ 75 mm from the tissue surface. TPM
images of the tissue sections obtained for 4000 s revealed the
AG2 distribution at depths of 75, 100, 125, and 150 mm, and
each image exclusively represents the distribution in a given
plane (Figure S8D and S9 in the Supporting Information).
TPM images were obtained up to a depth of 150 mm due to
the limited penetration of AG2 into the tissues during the
incubation; TPM images at deeper depths could be obtained
if the tissues were incubated for a longer period of time. As
expected, the uptake was most efficient near the tissue
surface; it decreased with larger imaging depths and was
negligible at a depth of > 150 mm. In addition, the uptake rate
was much slower than that in the cells, probably because of
the extracellular matrix, which is more abundant in aged
tissues. The sum of the TPEF intensities measured at depths
ranging from 75 to 150 mm was largest in the case of cancer
tissue, followed by cancer tissue treated with taxol, and then
normal tissue (Figure 4C). This result once again clearly
demonstrates the preferential uptake of AG2 by cancer tissue
and emphasizes the usefulness of AG2 in the diagnosis of
colon cancer.
for the diagnosis of colon cancer, for which we used images of
normal- and cancer-tissue slices from colon-cancer patients.
The bright-field image of the normal tissue clearly revealed
the presence of glands on the tissue surface. However, the
cancer tissue appeared almost amorphous and did not have a
definite structure (Figure 4A and Figure S8A in the Support-
ing Information). The tissues were incubated in artificial
cerebrospinal fluid (ACSF) for 4 h at 378C in the absence and
presence of taxol (50 mm), after which the AG2 uptake was
monitored by TPM by following the change in TPEF at a
depth of 100 mm. The result shows that AG2 uptake in the
cancer tissue is much faster than that in the normal tissue and
that the cancer tissue pretreated with taxol for 4 h exhibits
much slower AG2 uptake than the untreated one (Figure 4A
and B). A similar result was observed from the normal- and
cancer-tissue slices obtained immediately after colonoscopic
biopsy, which indicates that incubation of the tissue slices in
ACSF for 4 h did not significantly influence the relative
uptake rate (Figure S8B in the Supporting Information).
Furthermore, the uptake could be monitored for more than
3000 s without noticeable decay. These results strongly
support the applicability of AG2 in deep-tissue imaging for
the diagnosis of colon cancer, in addition to its useful
properties for in vivo imaging, namely high photostability
and low toxicity.
Colon cancer is known to originate from the mucosa in the
intestinal glands and spread into the interior of the tissue,[24]
so we have obtained TPM images at different depths from the
Finally, it is worthwhile to compare the near-infrared
(NIR) imaging with the IR dye 800CW-2DG[8] and the TPM
imaging with AG2. Both methods are capable of imaging
Figure 4. A) Images of normal tissue (a–c), cancer tissue (d–f), and cancer tissue treated with taxol (g–i). Normal tissues were incubated in ACSF
for 4 h, and cancer tissues were incubated in the absence and presence of taxol (50 mm) in ACSF for 4 h, after which AG2 uptake was monitored.
a, d, and g are bright-field images; b, e, and h are pseudocolored TPM images obtained after incubation with AG2 for 4000 s; and c, f, and i are
pseudocolored TPM images obtained after incubation with AG2 for 4 h. The TPM images were obtained at a depth of 100 mm by collecting the
TPEF spectra in the range of 520–620 nm on excitation with fs pulses at 780 nm. Scale bar: 30 mm. B) Time course of AG2 uptake by normal
tissue, cancer tissue, and cancer tissue treated with taxol (50 mm) at 100 mm depth as a function of time. C) Relative AG2 uptake by normal
tissue, cancer tissue, and cancer tissue treated with taxol (50 mm) for 4000 s. The columns indicate the sum of the TPEF intensities measured by
photomultiplier tube at depths of 75, 100, 125, and 150 mm from the tissue surface, relative to that of normal tissue. The data are the average of
three independent experiments.
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and S9 in the Supporting Information). This is not possible
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To conclude, we have developed a new TP tracer, AG2,
that can be excited by 780 nmfs laser pulses and can be easily
taken up by cancer cells and tissues through glucose-specific
translocation. AG2 is pH independent at a wide range of
physiological pH values (pH 4.0–10) and shows negligible
cytotoxicity and high photostability. It can monitor glucose
uptake in normal and colon-cancer tissues from human
patients for more than 3000 s and can visualize the efficacy
of anticancer agents in cancer cells and colon-cancer tissues at
depth of 75–150 mm by TPM. This compound may be useful
in diagonizing the early stages of cancer and in the develop-
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Received: March 3, 2009
Revised: August 4, 2009
Published online: September 18, 2009
Keywords: antitumor agents · fluorescent probes · glucose ·
.
imaging agents · two-photon microscopy
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