Development of novel nanocarrier-based near-infrared optical probes for in vivo tumor imaging

Article abstract of DOI:10.1007/s10895-011-1007-z

Optical imaging with near-infrared (NIR) fluorescent probes is a useful diagnostic technology for in vivo tumor detection. Our plan was to develop novel NIR fluorophoremicelle complex probes. IC7-1 and IC7-2 were synthesized as novel lipophilic NIR fluorophores, which were encapsulated in an amphiphilic polydepsipeptide micelle lactosome . The fluorophore-micelle complexes IC7-1 lactosome and IC7-2 lactosome were evaluated as NIR fluorescent probes for in vivo tumor imaging. IC7-1 and IC7-2 were synthesized and then encapsulated in lactosomes. The optical properties of IC7-1, IC7-2, IC7-1 lactosome and IC7-2 lactosome were measured. IC7-1 lactosome and IC7-2 lactosome were administered to tumor-bearing mice, and fluorescence images were acquired for 48 h. IC7-1 and IC7-2 were successfully synthesized in 12% and 6.3% overall yield, and maximum emission wavelengths in chloroform were observed at 858 nm and 897 nm, respectively. Aqueous buffered solutions of IC7-1 lactosome and IC7-2 lactosome showed similar fluorescence spectra in chloroform and higher or comparable quantum yields and higher photostability compared with ICG. Both lactosome probes specifically visualized tumor tissue 6 h post-administration. IC7-1 lactosome and IC7-2 lactosome could be promising NIR probes for in vivo tumor imaging. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s10895-011-1007-z

J Fluoresc (2012) 22:719–727
DOI 10.1007/s10895-011-1007-z
ORIGINAL PAPER
Development of Novel Nanocarrier-Based Near-Infrared
Optical Probes for In Vivo Tumor Imaging
Yoichi Shimizu & Takashi Temma & Isao Hara &
Ryo Yamahara & Ei-ichi Ozeki & Masahiro Ono &
Hideo Saji
Received: 5 September 2011 /Accepted: 18 October 2011 /Published online: 10 November 2011
#
Springer Science+Business Media, LLC 2011
.
.
.
Abstract Optical imaging with near-infrared (NIR) fluores-
cent probes is a useful diagnostic technology for in vivo tumor
detection. Our plan was to develop novel NIR fluorophore-
micelle complex probes. IC7-1 and IC7-2 were synthesized
as novel lipophilic NIR fluorophores, which were encapsu-
lated in an amphiphilic polydepsipeptide micelle “lacto-
some”. The fluorophore-micelle complexes IC7-1 lactosome
and IC7-2 lactosome were evaluated as NIR fluorescent
probes for in vivo tumor imaging. IC7-1 and IC7-2 were
synthesized and then encapsulated in lactosomes. The optical
properties of IC7-1, IC7-2, IC7-1 lactosome and IC7-2
lactosome were measured. IC7-1 lactosome and IC7-2
lactosome were administered to tumor-bearing mice, and
fluorescence images were acquired for 48 h. IC7-1 and IC7-2
were successfully synthesized in 12% and 6.3% overall
yield, and maximum emission wavelengths in chloroform
were observed at 858 nm and 897 nm, respectively. Aqueous
buffered solutions of IC7-1 lactosome and IC7-2 lactosome
showed similar fluorescence spectra in chloroform and
higher or comparable quantum yields and higher photo-
stability compared with ICG. Both lactosome probes
specifically visualized tumor tissue 6 h post-administration.
IC7-1 lactosome and IC7-2 lactosome could be promising
NIR probes for in vivo tumor imaging.
Keywords Optical Near-infrared Micelle Molecular
imaging
Introduction
Molecular imaging is an evolving field that is progressing
from basic research to use in clinical diagnosis [1, 2]. Among
several imaging methods, optical imaging can conveniently
and safely offer pronounced spatial and temporal resolution
that is desirable for in vivo applications, especially in the area
of cancer imaging. It is notable that near-infrared (NIR) light
(700–1,000 nm) transmits deeply through tissues with low
scattering, absorption, and auto-fluorescence [3, 4].
Although indocyanine green (ICG, Fig. 1a) is a NIR
probe approved by the Food and Drug Administration
(FDA) for monitoring cardiac function and hepatic output
[5–7], its low quantum yield [4] and low stability in
aqueous solution [3] are drawbacks to further applications
as an in vivo fluorescent molecular probe. ICG derivatives
with enhanced lipophilicity could help solve these problems
if they were to be utilized in a lipophilic environment, but
fluorescent tags for in vivo usage should be amenable to an
aqueous environment. On the other hand, a polydepsipep-
tide micelle composed of polysarcosine (PSar) and poly-L-
lactic acid (PLLA), named lactosome, has been reported to
be an in vivo stable, hydrophilic nanocarrier of ∼30 nm in
diameter, which can encapsulate hydrophobic compounds
and effectively deliver them into tumor tissue by an
enhanced permeability and retention (EPR) effect with
low nonspecific accumulation in the reticuloendothelial
system [8, 9]. Thus, our goal was to synthesize lipophilic
ICG derivatives possessing better fluorescence properties
and encapsulate them in lactosomes to provide NIR probes
for in vivo tumor imaging.
:
:
:
Y. Shimizu T. Temma M. Ono H. Saji (*)
Department of Patho-Functional Bioanalysis,
Graduate School of Pharmaceutical Sciences,
Kyoto University,
46-29 Yoshida Shimoadachi-cho, Sakyo-ku,
Kyoto 606-8501, Japan
e-mail: hsaji@pharm.kyoto-u.ac.jp
:
:
I. Hara R. Yamahara E.-i. Ozeki
Technology Research Laboratory, Shimadzu Corporation,
3-9-4 Soraku-gun, Seika-cho,
Kyoto 619-0237, Japan

720
J Fluoresc (2012) 22:719–727
Fig. 1 Chemical structures
of ICG (a), IC7-1 (c) and
IC7-2 (c)
To develop the novel lipophilic NIR compound, IC7-1
(Fig. 1b), a cyclohexenyl ring was introduced into the
polymethine chain of ICG to rigidify the structure and to
provide improved photostability and a higher quantum
yield [10, 11]. A related asymmetric ICG derivative
incorporating a quinoline moiety, IC7-2 (Fig. 1c), which
had an elongated emission spectrum, was also synthesized
for comparative purposes [12].
and the mixture was refluxed for 1 h. After cooling to rt, an
aniline/EtOH [1:1 (v/v), 18 mL] mixture was added
dropwise, and the reaction was continued at rt for 30 min.
The mixture was poured into ice cold H2O/HCl (10:1,
110 mL), and the resulting residue was filtered, washed
with cold H2O and THF, and then dried in vacuo to obtain
compound 1 (10.2 g, 53.6%); 1H NMR (DMSO-d6,
300 MHz) δ8.54 (s, 2H), 7.6-7.2 (m, 10H), 2.74 (t, 4H),
1.85 (m, 2H).
Materials and Methods
3-(5-Carboxy-pentyl)-1,1,2-trimethyl-1H-benz[e]indolium
iodide (2)
Materials
6-Bromohexanoic acid (8.4 g, 43.0 mmol) and potas-
sium iodide (7.2 g, 43 mmol) were dissolved in
toluene (5 mL). After addition of 1,1,2-trimethyl-1H-
benz[e]indole (3.0 g, 14.3 mmol), the mixture was
refluxed for 15 h. The resulting precipitate was washed
with THF, chilled H2O, and chloroform and then dried in
vacuo to obtain compound 2 (5.0 g, 77%); 1H NMR
(CD3OD) 8.33 (d, 1H, J=8.5), 8.24 (d, 1H, J=8.8), 8.16
(d, 1H, J=8.3), 8.02 (d, 1H, J=8.8), 7.8 (t, 1H, J=7.2),
7.71 (t, 1H, J=7.2), 4.64 (t, 2H, J=7.7), 2.35 (t, 2H, J=
6.9), 2.0-1.9 (m, 2H), 1.84 (s, 6H), 1.75-1.65 (m, 2H),
1.65-1.55 (m, 2H).
Aniline, cyclohexanone, N,N’-dicyclohexylcarbodiimide,
sodium hydride, 6-bromohexanoic acid, potassium iodide,
sodium acetate, 2-iodoethanol, N-hydroxysuccinimide, N,
N-dimethylformamide, and ethanol were purchased from
Wako Pure Chemical Industries, Ltd. 1,1,2-Trimethylbenz
[e]indole was purchased from Sekisui Medical CO, Ltd.
ICG was purchased from Tokyo Chemical Industry Co,
Ltd.
Instruments
UV–vis spectra were measured using a UV-1800 (SHI-
MADZU Corporation, Kyoto, Japan). Mass spectra were
acquired on a SHIMADZU LC-MS2010 EV (SHIMADZU
Corporation, Kyoto, Japan) and an Axima-CFR plus
(SHIMADZU Corporation, Kyoto, Japan). 1H-NMR spec-
tra were recorded on a JEOL ECP-300 (JEOL Ltd., Tokyo,
Japan). Fluorescence spectroscopy was performed with a
Fluorolog-3 (HORIBA Jobin Yvon Inc., Kyoto, Japan),
using a slit width of 5 nm for both excitation and emission
measurements.
2-[4′-Chloro-6′-(N-phenyl)-3′,5′-trimethyleneheptatrien-1-
yl]-1-(5-Carboxy-pentyl)-1,1,2-trimethyl-1H-benz[e]
indolium iodide (3)
A mixture of compound 1 (3.00 g, 8.35 mmol), 2 (3.77 g,
8.35 mmol) and anhydrous sodium acetate (0.753 g,
9.19 mmol) in anhydrous EtOH (75 mL) was refluxed for
6 h under a N2 atmosphere. After the reaction was
complete, the mixture was neutralized with 0.2 M phos-
phate buffer (pH 7.0), and then extracted with chloroform.
The extract was evaporated, and the residue was purified by
column chromatography to obtain compound 3 (1.45 g,
25.5%); 1H NMR (CD3OD) δ8.56 (s, 1H), 8.2-8.1 (m, 2H),
7.95-7.85 (m, 2H), 7.6-7.1 (m, 8H), 6.10 (d, 1H, J=13.8),
4.17 (t, 2H, J=7.2), 2.71 (t, 4H, J=5.8), 2.27 (t, 2H, J=7.4),
2.0-1.8 (m, 4H), 1.93 (s, 6H), 1.8-1.6 (m, 2H), 1.6-1.4 (m,
2H): MS (ESI, pos.) m/z calcd for C35H38ClN2O2 554
(M+), found 554.
Synthesis
N-[5-Anilino-3-chloro-2,4-(propane-1,3-diyl)-2,
4-pentadiene-1-ylidene]anilinium Chloride (1)
Phosphorus oxychloride (11 mL, 0.12 mol) was added
dropwise to anhydrous DMF (13 mL, 0.17 mol) at 0 °C.
After 1 h, cyclohexanone (5.5 mL, 0.053 mol) was added,

J Fluoresc (2012) 22:719–727
721
3-(2-Hydroxyethyl)-1,1,2-trimethyl-1H-benz[e]indolium
iodide (4)
20 min, and then N-hydroxysuccinimide (NHS, 246 mg,
2.14 mmol) was added. The solution was gradually allowed
to warm to room temperature, and it was stirred for 2 days.
After the reaction was complete, ethyl acetate was added to
the mixture, and the dicyclohexylurea was removed by
filtration. The solution was evaporated, and the residue was
purified by flash chromatography to obtain IC7-1-NHS
1,1,2-Trimethyl-1H-benz[e]indole (2.0 g, 9.556 mmol)
was dissolved in dry toluene (10 mL) at 80 °C under a
N2 atmosphere. To the solution was added 2-iodoethanol
(1.64 g, 9.556 mmol), and the mixture was refluxed for
2 h. After cooling to rt, a slightly blue residue was
collected, washed with toluene, and then dried in vacuo to
1
(100 mg, 14.9%); H NMR (CD3OD) δ8.50 (m, 2H), 8.23
(t, 2H, J=8.1), 8.1-7.9 (m, 4H), 7.7-7.4 (m, 6H), 6.45 (d,
1H, J=14.3), 6.24 (d, 1H, J=14.3), 4.41 (t, 2H, J=5.0),
4.24 (t, 2H, J=8.0), 4.03 (t, 2H, J=4.9), 2.8-2.6 (m, 6H),
2.78 (s, 4H), 2.0-1.5 (m, 8H), 2.01 (s, 6H), 1.99 (s, 6H):
MS calcd for C50H53ClN3O5 (M+) 811, found 811.
1
obtain compound 4 (1.21 g, 33%); H NMR (DMSO-d6)
δ8.39 (d, 1H, J=8.0), 8.29 (d, 1H, J=8.8), 8.22 (d, 1H, J=
8.0), 8.14 (d, 1H, J=9.1), 7.79 (t, 1H, J=7.2), 7.73 (t, 1H,
J=6.9), 4.72 (t, 2H, J=4.7), 3.94 (t, 2H, J=4.7), 2.93 (s,
3H), 1.78 (s, 6H).
2-[4′-Chloro-7′-(1″-(2-Hydroxyethyl)-4-
methylquinolinium)-3′,5′-trimethyleneheptatrien-1-yl]-1-(5-
Carboxypentyl)-1,1,2-trimethyl-1H-benz[e]indolium iodide
(IC7-2)
1-(2-Hydroxyethyl)-4-methylquinolinium iodide (5)
A mixture of 4-methylquinoline (1 g, 7 mmol) and 2-
iodoethanol (1.2 g, 7 mmol) in dry toluene (5 mL) was
stirred at 80 °C under a N2 atmosphere for 4 h. After
cooling, a yellow residue was collected, washed with
toluene, and then dried in vacuo to obtain compound 5
A mixture of compound 3 (414 mg, 0.608 mmol), 5
(210.7 mg, 0.669 mmol), and anhydrous sodium acetate
(54.9 mg, 0.669 mmol) in anhydrous EtOH (12 mL) was
refluxed for 8 h under a N2 atmosphere. After the reaction
was complete, the mixture was neutralized with 0.2 M
phosphate buffer (pH 7.0) and then extracted with chloro-
form and methanol. The organic extract was evaporated,
and the residue was purified by column chromatography to
1
(1.63 g, 74%); H NMR (CD3OD) δ9.14 (d, 1H, J=6.3),
8.62-8.52 (m, 2H), 8.25 (t, 1H, J=6.3), 8.06 (t, 1H, J=6.2),
7.97 (d, 1H, J=6.0), 5.14 (t, 2H, J=4.9), 4.08 (t, 2H, J=
5.0), 3.07 (s, 3H).
1
2-[4′-Chloro-7′-(1″-(2-Hydroxyethyl)-1,1,2-trimethyl-1H-
benz[e]indolium)-3′,5′-trimethyleneheptatrien-1-yl]-1-(5-
Carboxypentyl)-1,1,2-trimethyl-1H-benz[e]indolium iodide
(IC7-1)
obtain IC7-2 (221 mg, 46.7%); H NMR (CD3OD) δ8.65
(d, 1H, J=6.6), 8.57 (d, 1H, J=8.8), 8.39 (d, 1H, J=15.1),
8.26 (d, 1H, J=8.8), 8.15-7.65 (m, 7H), 7.44 (t, 1H, J=7.4),
7.35-7.1 (m, 3H), 5.69 (d, 1H, J=13.4), 4.05 (t, 2H, J=4.7),
3.83 (t, 2H, J=6.9), 2.8-2.65 (m, 4H), 2.21 (t, 2H, J=7.4),
1.90 (s, 6H), 2.0-1.6 (m, 7H), 1.6-1.2 (m, 3H): MS (ESI,
pos) m/z calcd for C41H44ClN2O3 (M+) 647, found 647.
A mixture of compound 3 (1.16 g, 1.70 mmol), 4 (0.714 g,
1.87 mmol), and anhydrous sodium acetate (0.153 g,
1.87 mmol) in anhydrous EtOH (29 mL) was refluxed for
5 h under a N2 atmosphere. After the reaction was
complete, the mixture was neutralized with 0.2 M phos-
phate buffer (pH 7.0) and then extracted with chloroform.
The organic extract was evaporated, and the residue was
purified by column chromatography to obtain IC7-1
IC7-2-NHS
To a solution of IC7-2 (355 mg, 0.457 mmol) and NHS
(157.7 mg, 1.370 mmol) in anhydrous DMF (3.5 mL) was
added DCC (188.5 mg, 0.914 mmol), and the mixture was
stirred for 1 h at 0 °C. The solution was allowed to
gradually warm to room temperature, and it was stirred for
2.5 days. After the reaction was complete, ethyl acetate was
added to the mixture, and the dicyclohexylurea was
removed by filtration. The filtrate was evaporated, and the
residue was purified by flash chromatography to obtain
1
(1.22 g, 85.3%); H NMR (CD3OD) δ8.54 (m, 2H), 8.26
(t, 2H, J=8.1), 8.05-7.95 (m, 4H), 7.7-7.4 (m, 6H), 6.46 (d,
1H, J=14.3), 6.28 (d, 1H, J=14.0), 4.42 (t, 2H, J=5.0),
4.28 (t, 2H, J=8.0), 4.03 (t, 2H, J=5.0), 2.75 (t, 4H, J=5.8),
2.31 (t, 2H, J=7.2), 2.1-1.8 (m, 6H), 2.05 (s, 6H), 2.01 (s,
6H), 1.8-1.6 (m, 2H), 1.6-1.4 (m, 2H): MS (ESI, pos) m/z
calcd for C46H50ClN2O3 714 (M+), found 714.
1
IC7-2-NHS (62 mg, 15.6%); H NMR (CD3OD) δ8.64 (d,
1H, J=6.6), 8.56 (d, 1H, J=8.5), 8.34 (d, 1H, J=12.4), 8.25
(d, 1H, J=8.8), 8.15-7.65 (m, 7H), 7.44(t, 1H, J=7.4), 7.35-
7.1 (m, 3H), 5.69 (br, 1H), 4.04 (t, 2H, J=4.7), 3.83 (br,
2H), 2.66 (s, 4H), 2.9-2.50 (m, 6H), 1.90 (s, 6H), 2.0-1.6
(m, 7H), 1.6-1.2 (m, 3H): MS (ESI, pos) m/z calcd for
C45H47ClN3O5 (M+) 744, found 744.
IC7-1-NHS
To a solution of IC7-1 (600 mg, 0.713 mmol) in anhydrous
DMF (12 mL) was added dicyclohexylcarbodiimide (DCC,
440 mg, 2.14 mmol) at 0 °C. The reaction was stirred for

722
J Fluoresc (2012) 22:719–727
Preparation of IC7-1 or IC7-2 encapsulated lactosomes
in H2O at 25 °C, following excitation at 815 nm and
820 nm, respectively. Quantum yields for IC7-1, IC7-1
lactosome, IC7-2, and IC7-2 lactosome were acquired by a
sphere accessory equipped with a Fluorolog-3 (HORIBA
Jobin Yvon Inc., Kyoto, Japan). Excitation spectra of IC7-
1 and IC7-2 (10 μM) were measured in methanol and
chloroform at 25 °C following emission at 880 nm and
900 nm, respectively, and those of IC7-1 lactosome and IC7-2
lactosome (1.3 μM) were measured in H2O at 25 °C
following excitation at 880 nm and 890 nm, respectively.
Absorption spectra of IC7-1 and IC7-2 (10 μM) were
measured in methanol and chloroform at 25 °C, and those
of IC7-1 lactosome and IC7-2 lactosome were measured in
water at 25 °C.
To a solution of IC7-1-NHS (1.1 mg, 1.68 μmol) or IC7-2-
NHS (1.0 mg, 1.68 μmol) in anhydrous DMF (1 mL) was
added PLLA30 bearing a free amino group (3.45 mg,
1.52 μmol). The solution was stirred at room temperature
overnight while shielding the reaction from light, and the
resulting mixture was purified by size exclusion chroma-
tography (Sephadex LH-20 column) using DMF as eluant.
The fraction of high molecular mass was collected and
dried in vacuo. IC7-1-PLLA: MS (MALDI-TOF, pos) m/z
calcd for C50H53ClN3O5 ([M+H] +) 2958, found 2958. IC7-
2-PLLA: MS (MALDI-TOF, pos) m/z calcd for
C
135H172ClN4O63 (M+) 2895, found 2895.
The amphiphilic polymer PLLA30-block-PSar70
The photostability of IC7-1 lactosome, IC7-2 lactosome,
and ICG were evaluated by a decrease of absorbance over
time. IC7-1 lactosome (1.3 μM in H2O), IC7-2 lactosome
(1.3 μM in H2O) and ICG (1.3 μM in H2O) were
continuously illuminated with a tungsten lamp in a
visible-ultraviolet spectrophotometer (UV-1800) for 1 h.
The absorption was measured at the corresponding wave-
lengths (IC7-1 lactosome: 830 nm, IC7-2 lactosome:
830 nm, and ICG: 780 nm) every 0.1 min for 1 h after
UV illumination was initiated.
(Fig. 2) was supplied by the Shimadzu Corporation. A
chloroform (1 mL) solution of the polymer (388 nmol) and
IC7-1-PLLA (3.9 nmol) or IC7-2-PLLA (3.9 nmol) was
dripped into a glass test tube. The solvent was removed
under reduced pressure to form a thin film on the walls of
the tube. PBS buffer (0.1 M, pH 7.4) was added to the test
tube, and the tube was heated at 82 °C for 20 min. The
resulting aqueous solution was filtered through a 0.20 μm
Acrodisc® syringe filter (Pall Corp, East Hills, NY). The
size distribution of IC7-1 lactosome and IC7-2 lactosome
was measured at 25 °C using a Zetasizer Nano-S90
(Malvern instruments Ltd., UK). The purities of IC7-1
lactosome and IC7-2 lactosome were measured by size
exclusion chromatography using a Superdex 200 10/300
GL column (GE Healthcare, U.K.) equilibrated with PBS
(−) at a flow rate of 0.5 ml/min. Absorbances at 215 nm
and 830 nm were used for detection of the lactosome and
IC7-1/IC7-2, respectively.
Tumor bearing mice
Female nude mice (BALB/c nu/nu) supplied by Japan SLC,
Inc. (Hamamatsu, Japan), were housed under a 12-h light/
12-h dark cycle and were given free access to food and
water. Animal experiments in this study were conducted in
accordance with institutional guidelines and were approved
by the Kyoto University Animal Care Committee, Japan.
FM3A cells were supplied by the Health Science Research
Resources Bank (HSRRB) (Osaka, Japan). The cell lines
were cultured in DMEM medium with 10% fetal bovine
serum at 37 °C in a humidified atmosphere containing 5%
CO2. FM3A cells, 5×106 cells in 100 μL of phosphate-
buffered saline (PBS), were subcutaneously inoculated into
the right hind legs of 7-week-old nude mice. Tumor bearing
mice 12 days after transplantation were used for the
imaging study.
Optical characteristics of IC7-1, IC7-1 lactosome, IC7-2
and IC7-2 lactosome
The log P values of IC7-1, IC7-2, and ICG were calculated by
ACD/Labs ver. 11.0 (Advanced Chemistry Development,
Inc., Toronto, Canada).
The fluorescence emission spectra of IC7-1 and IC7-2
(10 μM) were measured in methanol and chloroform at
25 °C, following excitation at 815 nm and 820 nm,
respectively. Fluorescence emission spectra of IC7-1
lactosome and IC7-2 lactosome (1.3 μM) were measured
In vivo imaging study
IC7-1 lactosome and IC7-2 lactosome (1.7 μM, 200 μL)
were injected into groups of three tumor-bearing mice, and
NIRF images were obtained using a Clairvivo OPT
(SHIMADZU Corporation, Kyoto, Japan) with a 785 nm
single laser (IC7-1 lactosome) or 808 nm single laser (IC7-
2 lactosome) for excitation and a 845/55 nm band-path
filter (IC7-1 lactosome) or 850 nm long-path filter (IC7-2
lactosome) for emission. During the imaging process, mice
Fig. 2 Chemical structure of the amphiphilic block polymer
(PLLA30-block-PSar70
)

J Fluoresc (2012) 22:719–727
723
were kept on the imaging stage under anesthesia using a
2.5% isoflurane gas in oxygen flow (1.5 L/min). For image
analysis, Clairvivo OPT measurement and display software
ver. 2.00 (SHIMADZU Corporation, Kyoto, Japan) was
used. Regions of interest (ROI) were designated for the
tumor and muscle (the left hind leg) on the back images and
for the liver on the front images. The average signal
intensity values were recorded for each ROI and were
plotted versus time.
Results
Synthesis of IC7-1, IC7-2, IC7-1 lactosome and IC7-2
lactosome
IC7-1 and IC7-2 were synthesized from cyclohexanone and
1,1,2-trimethyl-1H-benz[e]indole, as shown in Scheme 1, in
12% and 6.3% overall yield. The calculated log P values of
IC7-1 and IC7-2 were 3.62 and 0.90, respectively, while the
clog P of ICG was −1.69. To allow these fluorophores to
dissolve in aqueous solvent, they were encapsulated in
lactosomes using a film rehydration technique. The prepa-
ration of IC7-1 lactosome and IC7-2 lactosome was
confirmed by size exclusion chromatography using Super-
dex 200 10/300 GL (GE Healthcare, U.K.), and dual
absorbance peaks at 280 nm (lactosome) and 830 nm (IC7-
1/IC7-2) were only seen in the higher molecular weight
fraction (4–5 min). The particle sizes of IC7-1 lactosome
and IC7-2 lactosome were 33.4 3.1 nm (n=6) and 39.6
1.9 nm (n=4), respectively.
Statistics
Data are presented as means S.D. Statistical analysis of the
photostability of IC7-1 lactosome and IC7-2 lactosome vs
ICG was performed with two-way factorial ANOVA
followed by the Tukey-Kramer test. Statistical analyses
for fluorescence intensities of tumor vs muscle or liver of
mice administered IC7-1 lactosome or IC7-2 lactosome
were performed with two-way factorial ANOVA followed
by the Tukey-Kramer test.
Scheme 1 Synthetic scheme for the preparation of IC7-1, IC7-2 and NHS derivatives

724
J Fluoresc (2012) 22:719–727
Fluorescence characteristics of IC7-1, IC7-2, IC7-1
lactosome and IC7-2 lactosome
In vivo imaging study
Fluorescence images of tumor-bearing mice administered
IC7-1 lactosome or IC7-2 lactosome are shown in Fig. 5a
and b, respectively. The tumors of mice administered IC7-1
lactosome and IC7-2 lactosome were visible with both
agents 6 h after administration. The fluorescence intensity
at the tumor region of mice administered IC7-1 lactosome
(Fig. 5c) and IC7-2 lactosome (Fig. 5d) gradually increased
in a time-dependent manner reaching a peak 24 h after
administration that was significantly greater than the
fluorescence observed in the muscle and liver regions (p<
0.05). The fluorescence intensities of tumors 24 h after
probe administration were 3.1 fold (IC7-1 lactosome) and
2.5 fold (IC7-2 lactosome) greater than those at the 0 h time
point (immediately after probe administration). The inten-
sities in the muscle region (the opposite side from the
tumor) were relatively constant during the study, and the
tumor-to-muscle fluorescence ratios 24 h after administra-
tion were 2.2 0.7 (IC7-1 lactosome) and 2.2 0.2 (IC7-2
lactosome). The tumor-to-liver fluorescence ratios 24 h
after administration were 2.5 0.6 (IC7-1 lactosome) and
1.7 0.2 (IC7-2 lactosome), while the intensities in the liver
region of the IC7-2 lactosome group were transiently high
during the early imaging phase.
IC7-1, a lipophilic fluorophore with a cyclohexene ring in
the interior of the polymethine chain of the ICG structure,
had a maximum emission wavelength at 858 nm in
chloroform (Table 1). IC7-2 with an unsymmetrical cyanine
structure had a maximum emission wavelength at 897 nm
in chloroform, which as expected was a longer emission
wavelength compared with conventional NIR-dyes. In
addition to maximum emission wavelength, other fluores-
cent properties of IC7-1 and IC7-2 in chloroform such as
fluorescence spectrum, quantum yield, and extinction
coefficient were also quite similar to those of IC7-1
lactosome and IC7-2 lactosome measured in water (Fig. 3,
Table 1). In particular, IC7-1 and IC7-1 lactosome had
sharp absorbance and emission spectra and a narrow Stokes
shift, while IC7-2 and IC7-2 lactosome had broad absorp-
tion spectra and a relatively long Stokes shift.
The fluorescence characteristics of IC7-1, IC7-1 lacto-
some, IC7-2, and IC7-2 lactosome were compared with
ICG as summarized in Table 1. Both IC7-1 and IC7-2
displayed longer absorbance, excitation, and emission
wavelengths than ICG. IC7-1 and IC7-2 had higher
quantum yields and molar extinction coefficients in
chloroform than in methanol. IC7-1 lactosome had approx-
imately 52 nm and 43 nm longer maximum absorbance and
emission wavelengths and approximately 2.5 fold higher
quantum yield and molar extinction coefficients compared
with ICG. IC7-2 lactosome had a maximum emission
wavelength that was 55 nm longer than IC7-1 lactosome
and 98 nm longer than ICG.
Discussion
In this study, two lipophilic NIR fluorescent agents, IC7-1
and IC7-2, were prepared and evaluated (Fig. 1). To
enhance the utility of these agents for in vivo NIR optical
tumor imaging, they were encapsulated in a lactosome
formulation to take advantage of the ability of lactosomes
to effectively target and deliver lipophilic compounds to
tumor tissues. As expected, these fluorescent probes and
their lactosome complexes displayed absorption, excitation,
and emission spectra with red shifts (Fig. 3) as well as high
photostability compared with ICG (Fig. 4). The IC7-1
Regarding photostability of the three probes, the absor-
bances of IC7-1 lactosome and IC7-2 lactosome were stable
for 1 h under tungsten lamp irradiation (Fig. 4). On the
other hand, the absorbance of ICG significantly decreased
in a time-dependent manner (p<0.0001), and the normal-
ized absorbance of ICG after 60 min was 61% of the 0 min
value.
Table 1 Optical properties of IC7-1, IC7-1 lactosome, IC7-2, IC7-2 lactosome, and ICG
Entry
Solvent
Absmax
Exmax
Emmax
Quantum
yield
Extinction coefficient
(M−1 cm−1
)
IC7-1
CHCI3
MeOH
H2O
829
819
831
781
790
830
779
830
823
831
805
883
844
762
858
840
850
897
932
905
807
0.054
0.021
0.059
0.008
0.001
0.014
0.023
2.1×105
1.2×105
2.2×105
9.2×104
2.6×104
6.0×104
7.8×104
IC7-1 lactosome
IC7-2
CHCI3
MeOH
H2O
IC7-2 lactosome
ICG
H2O

J Fluoresc (2012) 22:719–727
725
Fig. 3 Emission (solid line), excitation (dotted line) and absorption
(dotted solid line) spectra of IC7-1 (a), IC7-2 (b), IC7-1 lactosome (c),
and IC7-2 lactosome (d). The spectra for IC7-1 and IC7-2 were
measured in chloroform and the spectra for IC7-1 lactosome and IC7-
2 lactosome were measured in water
lactosome possessed a 2.5 fold higher quantum yield than
ICG in aqueous solution (Table 1). In addition, IC7-1
lactosome and IC7-2 lactosome clearly allowed visualiza-
tion of tumor tissue 6 h post-administration and showed
significant fluorescence intensity in tumor tissue 24 h and
48 h after administration (Fig. 5).
In the design of IC7-1, a cyclohexene ring was
incorporated into the interior of the polymethine chain of
the ICG structure, because the added structural rigidity
provided by a ring would lead to a red-shift of the emission
and would increase the photostability and quantum yield in
a lipophilic environment [13]. As a result of this modifica-
tion, the emission spectra were elongated approximately
50 nm compared with ICG. IC7-2 was designed with
similar functional groups as IC7-1, but with an unsymmet-
rical structure, by replacing one of the hetero-tricyclic
moieties of IC7-1 with a quinoline ring, since it has
previously been reported that unsymmetrical fluorogenic
cyanines typically have longer wavelength emission spectra
[12, 14] than the corresponding symmetrical cyanines. As
expected, a longer emission wavelength (ca 900 nm) was
observed for IC7-2 compared to IC7-1, which could
provide superior properties for in vivo usage, although the
quantum yield of IC7-2 was somewhat lower than IC7-1.
Multicolor in vivo optical imaging is a promising
technique for detecting different tumor types or lymph
nodes simultaneously [15, 16]. Since NIR fluorescent
organic compounds that are commercially available or that
have been reported for in vivo optical imaging have
emission wavelengths in the range of 650–820 nm [17],
and widely used quantum dot based agents have toxicity
concerns due to associated heavy metals [18, 19], our
probes with emission spectra in the 800–900 nm range offer
a possible alternative for multicolor in vivo optical imaging.
To make IC7-1 and IC7-2 more effective for use in an
aqueous environment while maintaining their optical
properties, the fluorophores were encapsulated in nano-
Fig. 4 Photostability of IC7-1 lactosome (bold line), IC7-2 lactosome
(dotted bold line), and ICG (dotted line)

726
J Fluoresc (2012) 22:719–727
Fig. 5 a, b Back (upper) and front (lower) fluorescence images of
FM3A cell xenografted mice at 0 h (just after injection), 3, 6, 9, 24,
and 48 h after administration of IC7-1 lactosome (a) and IC7-2
lactosome (b). The arrows indicate the tumor (c, d). Fluorescence
intensities at the region of interest of the tumor, muscle, and liver of
mice administered IC7-1 lactosome (c) and IC7-2 lactosome (d). Data
are expressed as fluorescence intensity (mean S.D.). Comparisons of
the fluorescence intensities of tumor and muscle or liver were
performed with two-way factorial ANOVA followed by the Tukey-
Kramer test (*p<0.05 vs. muscle, **p<0.01 vs muscle, #p<0.05 vs.
liver)
carriers. As a nanocarrier for the imaging agents, we chose
an amphiphilic polydepsipeptide micelle “lactosome”.
Lactosome has a diameter of approximately 30–40 nm,
which is a suitable size for delivery to tumor tissue through
the EPR effect [20]. Furthermore, lactosomes have the
ability to escape the reticuloendothelial system (RES), and
thus, they are able to suppress nonspecific accumulation of
the probes in the abdominal region [21]. Experimental
results showed that IC7-1 lactosome and IC7-2 lactosome
had better quantum yields and photostability and longer
(more favorable for in vivo imaging usage) excitation/
emission wavelengths than ICG under aqueous conditions,
which suggested that the high lipophilicity of IC7-1 and
IC7-2 was adequate for encapsulation in lactosomes, i.e. the
hydrophobic core of the lactosome provided a suitable
environment for IC7-1 and IC7-2 to exert their favorable
optical properties. As clearly demonstrated in the in vivo
study, selective and pronounced signaling by the newly

J Fluoresc (2012) 22:719–727
727
5. Dzurinko VL, Gurwood AS, Price JR (2004) Intravenous and
indocyanine green angiography. Optometry 75(12):743–755
6. Landsman ML, Kwant G, Mook GA, Zijlstra WG (1976) Light-
absorbing properties, stability, and spectral stabilization of
indocyanine green. J Appl Physiol 40(4):575–583
developed NIR lactosome probes in tumors and low
background signals in liver and muscle tissue were
observed and semi-quantified. In addition, lactosome has
low toxicity since it is composed of biodegradable
substances, poly-sarcosine and poly-L-lactic acid [22, 23].
Therefore, IC7-1 lactosome and IC7-2 lactosome appear to
be promising, widely applicable nanocarrier probes for
noninvasive in vivo imaging techniques.
In this study, highly sensitive in vivo tumor imaging with
IC7-1 lactosome and IC7-2 lactosome by virtue of the EPR
effect was achieved. Regions of inflammation are known to
accumulate macromolecules since vascular permeability is
facilitated compared with normal tissues [24]; thus, these
probes certainly visualized such areas (data not shown).
Although tumor accumulation of a probe due to the EPR effect
is useful for detecting the presence of tumors and for treatment
by drugs carried within the probe, functional imaging that
targets functional molecules specifically expressed at the
lesion site could further enhance the specific diagnosis of
disease, a current trend toward personalization of patient
treatment. Thus, studies concerning the conjugation of
targeting moieties to the lactosome probes developed in this
study are now in progress.
7. Sakka SG (2007) Assessing liver function. Curr Opin Crit Care 13
(2):207–214
8. Makino A, Kizaka-Kondoh S, Yamahara R, Hara I, Kanzaki T,
Ozeki E, Hiraoka M, Kimura S (2009) Near-infrared fluorescence
tumor imaging using nanocarrier composed of poly(L-lactic acid)-
block-poly(sarcosine) amphiphilic polydepsipeptide. Biomaterials
30(28):5156–5160
9. Makino A, Yamahara R, Ozeki E, Kimura S (2007) Preparation of
novel polymer assemblies, “lactosome”, composed of Poly(L-
lactic acid) and poly(sarcosine). Chem Lett 36(10):1220–1221
10. Lee H, Mason JC, Achilefu S (2006) Heptamethine cyanine dyes
with a robust C–C bond at the central position of the chromophore. J
Org Chem 71(20):7862–7865
11. Strekowski L, Lipowska M, Patonay G (1992) Substitution
reactions of a nucleofugal group in heptamethine cyanine dyes.
Synthesis of an isothiocyanato derivative for labeling of proteins
with a near-infrared chromophore. J Org Chem 57(17):4578–
4580
12. Constantin TP, Silva GL, Robertson KL, Hamilton TP, Fague K,
Waggoner AS, Armitage BA (2008) Synthesis of new fluorogenic
cyanine dyes and incorporation into RNA fluoromodules. Org Lett
10(8):1561–1564
13. Ballou B, Ernst LA, Waggoner AS (2005) Fluorescence imaging
of tumors in vivo. Curr Med Chem 12(7):795–805
14. Mujumdar RB, Ernst LA, Mujumdar SR, Lewis CJ, Waggoner AS
(1993) Cyanine dye labeling reagents: sulfoindocyanine succinimidyl
esters. Bioconjug Chem 4(2):105–111
15. Kobayashi H, Koyama Y, Barrett T, Hama Y, Regino CA,
Shin IS, Jang BS, Le N, Paik CH, Choyke PL, Urano Y
(2007) Multimodal nanoprobes for radionuclide and five-color
near-infrared optical lymphatic imaging. ACS Nano 1(4):258–
264
16. Koyama Y, Barrett T, Hama Y, Ravizzini G, Choyke PL, Kobayashi
H (2007) In vivo molecular imaging to diagnose and subtype tumors
through receptor-targeted optically labeled monoclonal antibodies.
Neoplasia 9(12):1021–1029
17. Licha K (2002) Contrast agents for optical imaging. Top Curr
Chem 222:1–29
Conclusion
In this study, we synthesized IC7-1 lactosome and IC7-2
lactosome, which had suitable optical imaging properties
and achieved clear in vivo tumor imaging in mice. Results
from the application of IC7-1 lactosome and IC7-2
lactosome as in vivo imaging agents indicate that these
probes could be beneficial for the clinical detection of
tumors.
18. Derfus AM, Chan CWC, Bhatia SN (2004) Probing the
cytotoxicity of semiconductor quantum dots. Nano Lett 4(1):11–
18
19. Kirchner C, Liedl T, Kudera S, Pellegrino T, Munoz Javier A,
Gaub HE, Stolzle S, Fertig N, Parak WJ (2005) Cytotoxicity of
colloidal CdSe and CdSe/ZnS nanoparticles. Nano Lett 5(2):331–
338
Acknowledgement This study was supported by Grants-in-Aid for
Scientific Research from the Ministry of Education, Culture, Sports,
Science, and Technology, Japan. Part of this study was supported by
the New Energy and Industrial Technology Development Organization
(NEDO), Japan. We thank Dojindo Laboratories (Kumamoto, Japan)
for supporting the syntheses of IC7-1 and IC7-2.
20. Matsumura Y, Maeda H (1986) A new concept for macromolecular
therapeutics in cancer chemotherapy: mechanism of tumoritropic
accumulation of proteins and the antitumor agent smancs. Cancer
Res 46(12 Pt 1):6387–6392
21. Senior JH (1987) Fate and behavior of liposomes in vivo: a review
of controlling factors. Crit Rev Ther Drug Carrier Systems 3
(2):123–193
22. Gupta B, Revagadea N, Hilbornb J (2007) Poly(lactic acid) fiber:
an overview. Prog Polym Sci 32(4):455–482
23. Tsai G, Lane HY, Yang P, Chong MY, Lange N (2004) Glycine
transporter I inhibitor, N-methylglycine (sarcosine), added to
antipsychotics for the treatment of schizophrenia. Biol Psychiatry
55(5):452–456
References
1. Weissleder R, Pittet MJ (2008) Imaging in the era of molecular
oncology. Nature 452(7187):580–589
2. Willmann JK, van Bruggen N, Dinkelborg LM, Gambhir SS
(2008) Molecular imaging in drug development. Nat Rev Drug
Discov 7(7):591–607
3. Ntziachristos V, Ripoll J, Wang LV, Weissleder R (2005) Looking
and listening to light: the evolution of whole-body photonic
imaging. Nat Biotechnol 23(3):313–320
4. Weissleder R (2001) A clearer vision for in vivo imaging. Nat
Biotechnol 19(4):316–317
24. Maeda H, Wu J, Sawa T, Matsumura Y, Hori K (2000) Tumor
vascular permeability and the EPR effect in macromolecular
therapeutics: a review. J Control Release 65(1–2):271–284