Development of NIR fluorescent dyes based on Si-rhodamine for in vivo imaging

Article abstract of DOI:10.1021/ja210375e

We have developed a series of novel near-infrared (NIR) wavelength-excitable fluorescent dyes, SiR-NIRs, by modifying the Si-rhodamine scaffold to obtain emission in the range suitable for in vivo imaging. Among them, SiR680 and SiR700 showed sufficiently

Full text of DOI:10.1021/ja210375e

Communication
pubs.acs.org/JACS
Development of NIR Fluorescent Dyes Based on Si−rhodamine for in
Vivo Imaging
Yuichiro Koide,†,‡ Yasuteru Urano,§ Kenjiro Hanaoka,†,‡ Wen Piao,†,‡ Moriaki Kusakabe,∥,¶ Nae Saito,⊥
Takuya Terai,†,‡ Takayoshi Okabe,⊥ and Tetsuo Nagano*,†,‡
§
∥
⊥
†Graduate School of Pharmaceutical Sciences, Medicine, Agricultural and Life Sciences, and Open Innovation Center for Drug
Discovery, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
‡CREST, JST, Japan Science and Technology Agency, 3-5 Sanbancho, Chiyoda, Tokyo 102-0075, Japan
¶Matrix Cell Research Institute, Inc., Ushiku, Ibaraki, 300-1232, Japan
S
* Supporting Information
Group 14 rhodamines, containing silicon, germanium, or tin
ABSTRACT: We have developed a series of novel near-
infrared (NIR) wavelength-excitable fluorescent dyes, SiR-
NIRs, by modifying the Si−rhodamine scaffold to obtain
emission in the range suitable for in vivo imaging. Among
them, SiR680 and SiR700 showed sufficiently high
quantum efficiency in aqueous media. Both antibody-
bound and free dye exhibited high tolerance to photo-
bleaching in aqueous solution. Subcutaneous xenograft
tumors were successfully visualized in a mouse tumor
model using SiR700-labeled anti-tenascin-C (TN-C)
antibody, SiR700-RCB1. SiR-NIRs are expected to be
useful as labeling agents for in vivo imaging studies
including multicolor imaging, and also as scaffolds for NIR
fluorescence probes.
at the 10 position of the xanthene chromophore, show large
bathochromic shifts compared to the original rhodamines, but
still retain the advantages of the original rhodamines, including
high quantum efficiency in aqueous media (Φfl = 0.3−0.45),
tolerance to photobleaching, and sufficient water solubility for
biological applications.9−11 Although the emission maximum of
conventional Si−rhodamine (called SiR650 here; Figure 1),
Figure 1. Chemical structures of SiR650 and newly synthesized NIR
wavelength-excitable fluorescence dyes, SiR-NIRs.
one of the group 14 rhodamines, is as long as around 660 nm,
ear-infrared (NIR) (650−900 nm) wavelength-excitable
Nfluorescent dyes are attractive for biological applications this is not favorable for in vivo imaging. In view of the
wavelength dependence of the tissue transparency and the
requirement for sufficiently low background fluorescence, two
wavelength windows at 700 and 800 nm are often used as
emission channels for practical in vivo imaging.12−15 So, we set
out to develop novel NIR fluorescent dyes by modifying the
Si−rhodamine scaffold to obtain emission in the >700 nm
region required for practical in vivo imaging. Since it has been
reported that the emission wavelength of rhodamine can be
elongated by expansion of the xanthene ring, as seen in the
Alexa Fluor series,16 we applied this strategy to the Si−
rhodamine scaffold and obtained a series of NIR fluorescent
dyes, named SiR-NIRs. The structures of SiR680, SiR700, and
SiR720 are shown in Figure 1.
because of minimal tissue absorption and low background
autofluorescence from serum, proteins, and other biomolecules
in the NIR range, so that high contrast can be obtained
between target and background tissue.1,2 Consequently, NIR
fluorescent dyes have been applied for in vivo imaging to
diagnose or to elucidate the mechanisms of various diseases,
including cancer,3,4 atherosclerosis,5 rheumatoid arthritis,6 and
inflammation.7 Although many NIR light-excitable fluorescent
scaffolds have been reported, most of them do not emit
adequate fluorescence in aqueous media due to molecular
stacking or poor water-solubility, and the only widely used
fluorophore excitable in the NIR region is cyanine dye, as
exemplified by Cy5.5 and Cy7.3−7 However, even cyanine dyes
have some deficiencies for bioimaging; for example, in many
cases they suffer photobleaching.8 Accordingly, the develop-
ment of a novel fluorescent scaffold that is excitable in the NIR
region, photostable, and usable in aqueous media is still an
important challenge. Further, novel NIR fluorescent dyes with
favorable photophysical characteristics would be alternatives to
cyanine dyes as fluorescent labeling agents. Herein, we report
the development of a series of novel NIR wavelength-excitable
fluorescent dyes based on the rhodamine scaffold, and we
demonstrate their superior characteristics for in vivo
fluorescence imaging.
First, we examined the photophysical properties of SiR-NIRs
in aqueous media. As we had expected, all three dyes exhibited
absorption and emission maxima in the NIR region (670−740
nm) (Figure 2), and the fluorescence quantum efficiency
decreased as the wavelength became longer. It is noteworthy
that SiR680 and SiR700 exhibit sufficiently high fluorescence
quantum efficiency for application as NIR fluorescence dyes
(Φfl = 0.35 and 0.12) based on the comparison with Cy5.5 (Φfl
Received: November 4, 2011
Published: March 5, 2012
© 2012 American Chemical Society
5029
dx.doi.org/10.1021/ja210375e | J. Am. Chem. Soc. 2012, 134, 5029−5031

Journal of the American Chemical Society
Communication
Figure 2. Normalized absorption (left) and emission (right) spectra of
1 μM SiR-NIRs, measured in PBS at pH 7.4.
Figure 4. In vivo tumor imaging with SiR700-labeled anti-tenascin-C
antibody (SiR700-RCB1). Xenograft tumor model mice prepared with
human malignant meningioma HKBMM cells were intravenously
injected with 300 μL of 130 μg/mL SiR700-RCB1 (D/A = 1.6). The
tumor fluorescence images were obtained before and at 24 h after the
injection.
= 0.23), which is widely used as a NIR fluorescence labeling
agent (Table 1). We next examined their photostability. Cy5.5
showed relatively rapid photobleaching, but little decrease of
absorption and emission was observed in the cases of SiR680
and SiR700 (Figure 3A and Figure S10) and these SiR-NIRs
showed excellent tolerance to photobleaching. This result
suggests that SiR-NIRs may be suitable for various applications
requiring high photostability, including fluorescence imaging
with a high-power laser or repeated irradiation.
Table 1. Photophysical Properties of SiR-NIRs, Measured in
PBS at pH 7.4
a
dye
λabs (nm)
λfl (nm)
ε (M−1cm−1
)
Φfl
b
SiR650
SiR680
SiR700
SiR720
646
674
691
721
660
689
712
740
1.1 × 105
1.3 × 105
1.0 × 105
1.6 × 105
0.31
0.35
0.12
0.05
a
For determination of the fluorescence quantum efficiencies (Φfl),
Cy5.5 in PBS at pH 7.4 (Φfl = 0.23) was used as a fluorescence
standard.17 This value was taken from ref 9.
b
Figure 5. (A) Fluorescence images of frozen sections of tumors
harvested 3 days after intravenous injection of SiR700- or Cy5.5-
labeled anti-tenascin-C antibody (SiR700- or Cy5.5-RCB1). Dotted
squares were irradiated at 670 nm for 5 min. Fluoromount/Plus
(COSMO BIO) was used as a mounting medium for preparing the
slide samples. (B) In vivo imaging using a Pearl Impulse Imager with a
700 nm channel and an 800 nm channel. SiR700-RCB1 was iv injected
into a mouse xenograft tumor model prepared with human malignant
meningioma HKBMM cells at 24 h after the iv injection of IRDye800-
RCB1. The tumor was at the base of the right forefoot. SiR700-RCB1
(bottom row) and IRDye800-RCB1 (middle row) were separately
monitored in two wavelength windows, and merged images (800 nm:
green, 700 nm: red) are also shown (top row). Wavelength maxima for
excitation/emission filters were 685/720 nm (700 nm channel) for
SiR700, and 785/820 nm (800 nm channel) for IRDye800.
Fluorescence images were acquired at 24 h (left column), 48 h
(middle column) and 72 h (right column) after injection of IRDye800-
RCB1 and at 0 h (left column), 24 h (middle column) and 48 h (right
column) after injection of SiR700-RCB1 into the mouse xenograft
tumor model.
Figure 3. Photobleaching experiments. Normalized fluorescence
changes of 1 μM SiR680, SiR700 and Cy5.5 (A), and 0.3 μM
SiR700-RCB1 and Cy5.5-RCB1 (dye per antibody ratio (D/A) = 2.0)
(B) in PBS at pH 7.4 were measured during light irradiation (40 mW/
cm2, Cy5 filter) for 0, 10, 20, and 30 min.
Next, we prepared amine-reactive succinimidyl ester-bearing
SiR700, 2-Me-4-COOSu SiR700 (Figure 4A), for labeling of
biomolecules such as antibodies and proteins, and applied it for
in vivo tumor imaging targeted to the extracellular matrix
glycoprotein tanascin-C (TN-C), a glycoprotein known to be
ubiquitously expressed by malignant gliomas.18 For this
purpose, we prepared SiR700-labeled anti-TN-C-antibody
(SiR700-RCB1). Initially, we examined the tolerance of
SiR700-RCB1 to photobleaching. The photostability of anti-
body-bound SiR700 was extremely high (Figure 3B), being
similar to that of free SiR700 in aqueous solution. We then
intravenously injected SiR700-RCB1 into a mouse xenograft
tumor model prepared with human malignant meningioma
HKBMM cells. The fluorescence signal of SiR700 in the tumor
was clearly observed at 24 h after the injection, and remained
observable for more than 10 days (Figure 4B). Observation of
the frozen tumor sections confirmed that the fluorescence was
localized in the extracellular matrix (Supporting Figure S1), in
accordance with fluorescence microscopic images presented
previously.19,20 We also examined the photostability of SiR700
in the frozen sections. Four commercially available mounting
media containing an antifading agent were used to prepare slide
samples. In two cases, the fluorescence of Cy5.5 faded
significantly during continuous scanning for 5 min by a
confocal microscope operating at 670 nm, whereas no fading of
SiR700 was observed in any of the media (Figure 5A and
Supporting Figures S2−S5). This result indicates that SiR700 is
5030
dx.doi.org/10.1021/ja210375e | J. Am. Chem. Soc. 2012, 134, 5029−5031

Journal of the American Chemical Society
Communication
(5) Faust, A.; Waschkau, B.; Waldeck, J.; Holtke, C.; Breyholz, H. J.;
̈
more suitable than Cy5.5 for longer-term observation under
high-power laser irradiation. To examine further the practical
usefulness of SiR700, we conducted in vivo experiments using
two kinds of dye-labeled RCB1, SiR700-RCB1 and IRDye800-
RCB1. The N-hydroxysuccinimide (NHS) ester of IRDye
800CW was purchased from LI-COR Bioscience (Lincoln,
NE). One conjugate was injected at 24 h after injection of the
other conjugate, followed by observation of the fluorescence in
the tumor region using two wavelength windows (700 nm for
SiR700, and 800 nm for IRDye800). The tumor region was well
imaged with a high signal-to-noise ratio by using either SiR700-
RCB1 or IRDye800-RCB1. Further, the fluorescence of
SiR700-RCB1 and IRDye800-RCB1 in the tumor could be
separately monitored (Figure 5B), so that multicolor imaging
with these dyes is feasible.
In summary, we have developed a series of novel NIR
wavelength-excitable fluorescent dyes, SiR-NIRs, by modifying
the Si−rhodamine scaffold to obtain longer-wavelength
emission in the range suitable for in vivo imaging. Among
them, SiR680 and SiR700 showed sufficiently high quantum
efficiency in aqueous media for fluorescence imaging, and
exhibited high tolerance to photobleaching in the free or
protein-bound state in aqueous solution. This characteristic is
favorable for prolonged observation, and we also confirmed
that the fluorescence remained observable after prolonged
storage of frozen sections (for at least 3 months; Supporting
Figures S6−S9). Further, subcutaneous xenograft tumors in a
mouse tumor model were visualized with SiR700-labeled anti-
TN-C antibody. SiR-NIRs are expected to be useful as labeling
tools for a wide range of in vivo imaging studies including
multicolor imaging, and also as a novel scaffold for NIR
fluorescence probes. Development of NIR fluorescence probes
based on these dyes is in progress.
Wagner, S.; Kopka, K.; Heindel, W.; Schafers, M.; Bremer, C.
̈
Bioconjugate Chem. 2008, 19, 1001−1008.
(6) Gompels, L. L.; Madden, L.; Lim, N. H.; Inglis, J. J.; McConnell,
E.; Vincent, T. L.; Haskard, D. O.; Paleolog, E. M. Arthritis Rheum.
2011, 63, 107−117.
(7) Sheth, R. A.; Tam, J. M.; Maricevich, M. A.; Josephson, L.;
Mahmood, U. Radiology 2009, 251, 813−821.
(8) Song, F.; Peng, X.; Lu, E.; Zhang, R.; Chen, X.; Song, B. J.
Photochem. Photobiol., A 2004, 168, 53−57.
(9) Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T. ACS
Chem. Biol. 2011, 6, 600−608.
(10) Fu, M.; Xiao, Y.; Qian, X.; Zhao, D.; Xu, Y. Chem. Commun.
2008, 15, 1780−1782.
(11) Koide, Y.; Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T. J. Am.
Chem. Soc. 2011, 133, 5680−5682.
(12) Hawrysz, D. J.; Sevick-Muraca, E. M. Neoplasia 2000, 2, 388−
417.
(13) Troyan, S. L.; Kianzad, V.; Gibbs-Strauss, S. L.; Gioux, S.;
Matsui, A.; Oketokoun, R.; Ngo, L.; Khamene, A.; Azar, F.; Frangioni,
J. V. Ann. Surg. Oncol. 2009, 16, 2943−2952.
(14) Frangioni, J. V. Curr. Opin. Chem. Biol. 2003, 7, 626−634.
(15) Shu, X.; Royant, A.; Lin, M. Z.; Aguilera, T. A.; Lev-Ram, V.;
Steinbach, P. A.; Tsien, R. Y. Science 2009, 324, 804−807.
(16) Haugland, R. P., Ed. A Guide to Fluorescent Probes and Labeling
Technologies, 10th ed.; Molecular Probes, Inc.: Eugene, OR, 2005.
(17) Mujumdar, S. R.; Mujumdar, R. B.; Grant, C. M.; Waggoner, A.
S. Bioconjugate Chem. 1996, 7, 356−362.
(18) Orend, G.; Chiquet-Ehrismann, R. Cancer Lett. 2006, 244, 143−
163.
(19) Hicke, B. J.; Stephens, A. W.; Gould, T.; Chang, Y. F.; Lynott, C.
K.; Heil, J.; Borkowski, S.; Hilger, C. S.; Cook, G.; Warren, S.;
Schmidt, P. G. J. Nucl. Med. 2006, 47, 668−678.
(20) Koyama, Y.; Kusubata, M.; Yoshiki, A.; Hiraiwa, N.; Ohashi, T.;
Irie, S.; Kusakabe, M. J. Invest. Dermatol. 1998, 111, 930−935.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis, experimental details and characterization of SiR-
NIRs, and results of imaging experiments in a mouse tumor
model. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
tlong@mol.f.u-tokyo.ac.jp
■
Notes
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 22000006 to T.N., and Grant
Nos. 20117003 and 19205021 to Y.U.), and by the Industrial
Technology Development Organization (NEDO) of Japan
(08007568-0 to T.N. and 08007408-0 to M.K.).
REFERENCES
■
(1) Weissleder, R.; Ntziachristos, V. Nat. Med. 2003, 9, 123−128.
(2) Weissleder, R. Nat. Biotechnol. 2001, 19, 316−317.
(3) Becker, A.; Hessenius, C.; Licha, K.; Ebert, B.; Sukowski, U.;
Semmler, W.; Wiedenmann, B.; Grotzinger, C. Nat. Biotechnol. 2001,
̈
19, 327−331.
(4) Ogawa, M.; Kosaka, N.; Choyke, P. L.; Kobayashi, H. Cancer Res.
2009, 69, 1268−1272.
5031
dx.doi.org/10.1021/ja210375e | J. Am. Chem. Soc. 2012, 134, 5029−5031