Near-infrared-fluorescence imaging of lymph nodes by using liposomally formulated indocyanine green derivatives

Article abstract of DOI:10.1016/j.bmc.2013.12.026

Liposomally formulated indocyanine green (LP-ICG) has drawn much attention as a highly sensitive near-infrared (NIR)-fluorescence probe for tumors or lymph nodes in vivo. We synthesized ICG derivatives tagged with alkyl chains (ICG-Cn), and we examined NIR-fluorescence imaging for lymph nodes in the lower extremities of mice by using liposomally formulated ICG-Cn (LP-ICG-Cn) as well as conventional liposomally formulated ICG (LP-ICG) and ICG. Analysis with a noninvasive preclinical NIR-fluorescence imaging system revealed that LP-ICG-Cn accumulates in only the popliteal lymph node 1 h after injection into the footpad, whereas LP-ICG and ICG accumulate in the popliteal lymph node and other organs like the liver. This result indicates that LP-ICG-Cn is a useful NIR-fluorescence probe for noninvasive in vivo bioimaging, especially for the sentinel lymph node.

Full text of DOI:10.1016/j.bmc.2013.12.026

Bioorganic & Medicinal Chemistry 22 (2014) 721–727
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Near-infrared-fluorescence imaging of lymph nodes by using
liposomally formulated indocyanine green derivatives
Taro Toyota a,b,c, Hiromichi Fujito , Akiko Suganami , Tomoki Ouchi , Aki Ooishi , Akira Aoki ,
d
b
e
e
f
f
f
f
a
b
Kazutaka Onoue , Yutaka Muraki , Tomoyuki Madono , Masanori Fujinami , Yutaka Tamura ,
g,h,
⇑
Hideki Hayashi
a
Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
Department of Bioinformatics, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
Department of Basic Science, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan
Department of Medical System Engineering, Faculty of Engineering, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
Division of Nanoscience, Graduate School of Advanced Integration Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
Yamada Chemical Co. Ltd, 1-1 Kamichoshi-cho, Kamitoba, Minami-ku, Kyoto 601-8105, Japan
Center for Frontier Medical Engineering, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan
Department of Frontier Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
b
c
d
e
f
g
h
a r t i c l e i n f o
a b s t r a c t
Article history:
Liposomally formulated indocyanine green (LP-ICG) has drawn much attention as a highly sensitive near-
infrared (NIR)-fluorescence probe for tumors or lymph nodes in vivo. We synthesized ICG derivatives
tagged with alkyl chains (ICG-Cn), and we examined NIR-fluorescence imaging for lymph nodes in the
lower extremities of mice by using liposomally formulated ICG-Cn (LP-ICG-Cn) as well as conventional
liposomally formulated ICG (LP-ICG) and ICG. Analysis with a noninvasive preclinical NIR-fluorescence
imaging system revealed that LP-ICG-Cn accumulates in only the popliteal lymph node 1 h after injection
into the footpad, whereas LP-ICG and ICG accumulate in the popliteal lymph node and other organs like
the liver. This result indicates that LP-ICG-Cn is a useful NIR-fluorescence probe for noninvasive in vivo
bioimaging, especially for the sentinel lymph node.
Received 7 October 2013
Revised 5 December 2013
Accepted 7 December 2013
Available online 20 December 2013
Keywords:
Liposome
Indocyanine green (ICG)
Near-infrared (NIR)-fluorescence probe
Phospholipid
Ó 2013 Elsevier Ltd. All rights reserved.
Lymph node
1
. Introduction
In the biological and medical sciences, noninvasive imaging
characteristics, but more importantly, because it was approved
by the Food and Drug Administration in the United States.63 SLNs
are, hypothetically, the first nodes to receive drainage from pri-
mary tumors, and detection of these during operation could pre-
vent morbidity of surgery, improve lymph node staging, and thus
contribute to survival and quality of life after surgery. Thus far,
SLN mapping procedures with ICG have been tested in various
types of malignancies and found to be equally useful and less cum-
1–4
technologies such as radioactive isotope imaging,
echo imaging,
used. Among these, near-infrared (NIR) fluorescence imaging has
drawn much attention because of low tissue autofluorescence
and deep penetration of light into tissues at wavelengths between
ultrasonic
5
–7
8–10
and nuclear magnetic resonance imaging
are
1
1–18
6
50 and 900 nm.
bersome than conventional techniques using radioactive colloids
Both NIR-fluorescent dyes19–28 and colloidal particles29–40 are
or dyes.
60–62,64–67
However, one of the issues to be considered
commonly used in biomedical analyses, and NIR-fluorescent carbo-
cyanine dyes are of great interest because of their ease of synthesis,
biocompatibility, tunable spectral properties, and exceptionally
high molar absorptivity in the NIR region; therefore, they have
been extensively used in cellular and animal imaging applica-
while using this technique is the rapid diffusion characteristic of
this dye. Wide blurring of the dye around injection sites and rapid
migration of the dye beyond the SLN have been reported, and these
could hamper the accuracy and simplicity of detecting SLNs with
this technique.
4
1–59
tions.
Indocyanine green (ICG) is one of such NIR-fluorescent
Liposomes, which are closed bilayer membranes composed of
phospholipids, have traditionally been of interest to medical
researchers for use as drug carriers and recently have been used
as colloidal probes for bioimaging because their size allows for
dyes, and it has recently garnered much attention in detecting
sentinel lymph nodes (SLNs)6
0–62
owing to its abovementioned
6
8–76
the targeting of a specific region of tumor.
As a result,
⇑
Corresponding author. Tel.: +81 43 290 3114; fax: +81 43 290 3403.
E-mail address: hhayashi@faculty.chiba-u.jp (H. Hayashi).
liposomally formulated ICG (LP-ICG) has been recently considered
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.026

7
22
T. Toyota et al. / Bioorg. Med. Chem. 22 (2014) 721–727
the ideal candidate as an NIR-fluorescence imaging probe. For in-
stance, Jeong et al. have reported the modification of liposome
via mannosylation of phospholipid and the lymph node imaging
74
N
by ICG contained in the modified liposome. Proulx et al. have
reported on the high stability and high sensitivity of LP-ICG for
NIR-fluorescence imaging of lymph nodes in vivo by its adminis-
tration to lower extremities of mice.75 However, LP-ICG has still
shown to accumulate in not only a first draining lymph node but
also a second-tier node in the lower extremities of mice. We
hypothesized that ICG can leak out from the liposomal membrane
in vivo; thus, we synthesized a novel ICG, which is more hydropho-
bic than conventional ICG, by substitution of one sulfonate groups
of the ICG with an alkyl chain (ICG-Cn; n = 4, 6, 8, 10, and 18). We
examined the spectral properties of ICG-Cn in both an organic solu-
tion and a liposomal dispersion, and we visualized the lymph
nodes in the lower extremities of mice by using liposomally formu-
lated ICG-Cn (LP-ICG-Cn) as well as LP-ICG and ICG under a
NIR-fluorescence imaging system.
(iii)
(i)
N
N
(
CH
2
)
4
I
R
3
O S
(
ii)
O
2
. Material and methods
(
iv)
N
N
(
2 4
CH )
2
1
.1. Synthesis of 18-(2-((1E,3E,5E,7E)-7-(1,1-dimethyl-3-butyl-
H-benzo[e]indol-2(3H)-ilydene)hepta-1,3,5-trienyl)-1,1-
3
O S
dimethyl-1H-benzo[e]indolium-3-yl)octadecane-1-sulforic acid
(
ICG-C18) and other ICG-Cn (n = 4, 6, 8, and 10)
The process of ICG-C18 synthesis follows the conventional pro-
cesses for synthesis of ICG and other ICG derivatives reported else-
N
N
4
5,56,77
where (Scheme 2).
2,3,3-Trimethyl-4,5-benzo-3H-indole
(
CH
2
)
4
R
(
3.1 g, 15 mmol) and 1,4-butane sultone (2.1 g, 15 mmol) were
3
O S
mixed in a round-bottomed flask (25 mL) under a nitrogen atmo-
sphere, and the reaction solution was stirred at 80 °C for 4 h and
then cooled at room temperature. By addition of acetone to the
reaction mixture, the residue was dissolved, and the product was
crystallized. The reaction product, 2,3,3-trimethyl-1-(sulfobutyl)-
ICG-Cn
Scheme 2. Synthesis of ICG-Cn: (i) 1,4-butane sultone, (ii) glutacon-aldehyde dianil
hydrogen chloride salt, (iii) C
pyridine.
n
H2n+1I (n = 4, 6, 8, 10, and 18), 2-butanone, and (iv)
4
,5-benzoindolium inner salt, was separated by filtration and
of ethyl acetate (40 mL) to crystallize the product, 1-octadecyl-
,3,3-trimethyl-4,5-benzo[e]indolium iodide, was separated by
filtration and rinsed twice with ethyl acetate. The product was
obtained as a gray crystal (4.4 g, 19%).
rinsed with acetone. The product was obtained as a gray crystal
2
(
1.17 g, 23%).
A reaction mixture of the gray crystal (1.04 g, 3.0 mmol) and
glutaconaldehyde dianil hydrogen chloride salt (0.94 g, 3.3 mmol)
was stirred at 120 °C for 1 h and then cooled at room temperature
to crystallize the product, 2-(6-acetanilido-1,3,5-hexatrienyl)-3,3-
dimethyl-1-(sulfobutyl)-4,5-benzo[e]indolium inner salt. The salt
was then separated by filtration and rinsed with acetone. The prod-
uct was obtained as a dark purple crystal (0.97 g, 58%).
Finally, a pyridine solution (16 mL) of the indolium inner salt
(
1.58 g, 3.0 mmol) and the indolium iodide (1.77 g, 3.0 mmol)
was stirred at 50 °C for 1 h under a nitrogen atmosphere. After
cooling at room temperature and addition of distilled water
(
40 mL), the crystallized product was separated by filtration and
dissolved in ethyl acetate. The residue of the solution was filtered
and recrystallized in 40 mL of chloroform/ethyl acetate (1/1, vol-
ume ratio), and the product was obtained as a dark green crystal
A 2-butanone solution (40 mL) of 2,3,3-trimethyl-4,5-benzo-
3H-indole (8.4 g, 40 mmol) and 1-iodooctadecane (16.8 g,
44 mmol) was stirred in a round-bottomed flask (100 mL) at
70 °C for 18 h and then cooled at room temperature. After addition
(
1
1.39 g, 53%). The other ICG derivatives: ICG-Cn (n = 4, 6, 8, and
0), with different alkyl chains from ICG-C18, were synthesized
using the same procedure and the corresponding indolium salts.
Yields of 46% (ICG-C4), 59% (ICG-C6), 57% (ICG-C8), and 44%
(
ICG-C10) were obtained.
The H NMR spectra of ICG-Cn (n = 4, 6, 8, 10, and 18) were run
1
6
at 300 MHz in DMSO-d by using a Lambda-300 NMR spectrometer
N
N
(JEOL, Tokyo, Japan). The infrared spectra of these compounds were
recorded on an IRPrestige-21 spectrometer (Shimadzu, Kyoto, Ja-
(
CH
2
)
4
R
À1
pan) in the range of 4000–400 cm using potassium bromide pel-
3
O S
lets. Electron spray ionization mass spectroscopy (ESI-MS) was
R:
C
4
H
8
SO
3
Na
ICG
examined using LCMS-2010EV (Shimadzu, Kyoto, Japan).
C
C
4
H
9
ICG-C4
1
ICG-C4: H NMR (300 MHz, DMSO-d
6
): d 0.94 (t, 3H, J = 7.0 Hz);
6
H
13
17
ICG-C6
ICG-C8
ICG-C10
1
1
.43 (m, 2H); 1.79 (m, 18H); 2.54 (m, 2H); 4.20 (m, 4H); 6.36 (d,
C
C
8
H
H, J = 13.8 Hz); 6.57 (m, 3H); 7.47 (m, 2H); 7.63 (m, 3H); 7.78
10
18
H
21
37
À1
(
m, 2H); 7.99 (m, 6H); 8.22 (d, 2H, J = 8.5 Hz). IR (KBr, cm ):
C
H
ICG-C18
3
051, 2930, 2871, 1630, 1418, 1310, 1138. ESI-MS (methanol/ace-
+
Scheme 1. Structures of ICG and ICG-Cn (n = 4, 6, 8, 10, and 18).
tonitrile = 1:1): m/z = 673 [M+H ].

T. Toyota et al. / Bioorg. Med. Chem. 22 (2014) 721–727
723
1
ICG-C6: H NMR (300 MHz, DMSO-d
.20–1.45 (m, 6H); 1.65–2.0 (m, 18H); 2.42–2.65 (m, 2H); 4.20
quintet, 4H); 6.37 (d, 1H, J = 12.7 Hz); 6.61 (m, 3H); 7.47 (m,
6
): d 0.85 (t, 3H, J = 7.0 Hz);
separated from the liposomal dispersion using an absorbance spec-
trophotometer at 805 nm (UV-1800, Shimadzu, Kyoto, Japan).
1
(
2
8
1
H); 7.59–7.70 (m, 3H); 7.72–7.85 (m, 2H); 7.90–8.07 (m, 6H);
.22 (d, 2H, J = 8.5 Hz). IR (KBr, cm ): 3051, 2930, 1630, 1508,
2.3. Absorbance and fluorescence spectroscopy
À1
417, 1354, 1094, 924, 665. ESI-MS (methanol/acetonitrile = 1:1):
The absorbance spectrophotometer was also used to measure
the absorption spectra of ICG and ICG-Cn dissolved in a spectro-
scopic grade ethanol (Kanto, Tokyo, Japan). Excitation and emission
spectra of ICG, ICG-Cn, LP-ICG, and LP-ICG-Cn were obtained using
a fluorospectrometer (FP-6600; JASCO, Japan). Spectroscopic grade
chloroform (Kanto) was used as the solvent for ICG and ICG-Cn.
+
m/z = 701 [M+H ].
1
ICG-C8: H NMR (300 MHz, DMSO-d
6
): d 0.82 (t, 3H, J = 6.6 Hz);
1
1
2
2
.32 (m, 10H); 1.79 (m, 18H); 2.54 (m, 2H); 4.20 (m, 4H); 6.36 (d,
H, J = 13.7 Hz); 6.57 (m, 3H); 7.48 (m, 2H); 7.64 (m, 3H); 7.78 (m,
À1
H); 7.99 (m, 6H); 8.22 (d, 2H, J = 8.6 Hz). IR (KBr, cm ): 3051,
928, 2855, 1624, 1418, 1310, 1138. ESI-MS (methanol/acetoni-
+
trile = 1:1): m/z = 729 [M+H ].
2.4. NIR-fluorescence imaging
1
ICG-C10: H NMR (300 MHz, DMSO-d
6
): d 0.81 (t, 3H, J = 6.7 Hz);
1
1
2
2
.32 (m, 14H); 1.80 (m, 18H); 2.54 (m, 2H); 4.20 (m, 4H); 6.36 (d,
To obtain the NIR-fluorescence image of the liposome disper-
sions after injection into the mice, a noninvasive fluorescence
imaging system (IVIS Lumina II; Caliper, MA, USA) was used with
an XFL-HR Fluorescence Filter Option High Range (720–840 nm)
detection filter. The fluorescence photons from each sample were
counted and statistically compared using a t-test, with statistical
significance set at p < 0.05.
H, J = 13.7 Hz); 6.57 (m, 3H); 7.47 (m, 2H); 7.63 (m, 3H); 7.78 (m,
À1
H); 7.99 (m, 6H); 8.22 (d, 2H, J = 8.6 Hz). IR (KBr, cm ): 3060,
924, 2853, 1635, 1418, 1310, 1138. ESI-MS (methanol/acetoni-
+
trile = 1:1): m/z = 757 [M+H ].
1
ICG-C18: H NMR (300 MHz, DMSO-d
6
): d 0.82 (t, 3H, J = 6.6 Hz);
1
1
2
2
.30 (m, 30H); 1.80 (m, 18H); 2.54 (m, 2H); 4.20 (m, 4H); 6.36 (d,
H, J = 13.8 Hz); 6.56 (m, 3H); 7.47 (m, 2H); 7.63 (m, 3H); 7.77 (m,
À1
H); 7.99 (m, 6H); 8.22 (d, 2H, J = 8.1 Hz). IR (KBr, cm ): 3040,
2.5. Injection of ICG solution or liposomal dispersion into
footpads of mice
922, 2851, 1624, 1414, 1310, 1136. ESI-MS (methanol/acetoni-
+
trile = 1:1): m/z = 869 [M+H ].
Six-week-old male KSN mice were purchased from Japan SLC,
Inc. (Shizuoka, Japan) and kept under specific pathogen-free and
humane conditions in the animal care facility of the Research Cen-
ter for Frontier Medical Engineering, Chiba University, Japan. After
anesthetization by intraperitoneal injection of pentobarbital
2
.2. Preparation of LP-ICG and LP-ICG-Cn
1
,2-Dioleoyl-3-sn-glycero-phosphocholine (10.0 mM; NOF,
Tokyo, Japan), cholesterol (1.0 mM; NOF), phosphatidylethanol-
amine-N-methoxy-polyethylene
glycol(5000)-dioleoyl-glycero
(40 mg/kg; Dainippon Sumitomo Pharma, Japan), 10 lL of ICG
À1
ammonium salt (5.0 Â 10 mM; NOF), and ICG or ICG-Cn were
solution, LP-ICG, or LP-ICG-C18 dispersion was injected into a rear
footpad of the mouse. The stage beneath the mouse was kept at
30 °C during the imaging. The animal experiment protocol was re-
viewed and approved by the Institutional Animal Care and Use
Committee of Animal Experimentation, Chiba University.
dissolved in an mixed organic solvent of chloroform/methanol
(
9:1, volume ratio). A thin lipid film was formed by removal of
the solvent under reduced pressure. After addition of the glucose
isotonic solution (5% [w/v]) at room temperature, the liposomal
dispersion was filtered through a 0.4-lm pore filter made of poly-
carbonate (Millipore, MA, USA). The liposomal size distribution
2.6. Single-dose toxicity assay of LP-ICG-C18
was determined by dynamic light scattering measurements
(
UPA-UT151; NIKKISO, Tokyo, Japan).
To determine the ratio of ICG or ICG-C18 leaking from the lipo-
All animal procedures were approved by Chiba University and
the Kamakura Techno-Science Inc. Institutional Animal Care and
Use Committee. Six-week-old male (SPF, Crl:CD [SD]) rats were
purchased from Charles River Laboratories Japan (Kanagawa,
Japan) and housed separately under specific pathogen-free and
somes, we used a preparative gel permeable chromatographic col-
umn (PD-10; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and
measured the absorbance of the obtained fraction (200 lL)
Figure 1. Fluorescence excitation (a) and emission (b) spectra of ICG-Cn dissolved in chloroform (32 lM).

7
24
T. Toyota et al. / Bioorg. Med. Chem. 22 (2014) 721–727
clinically useful NIR-fluorescent molecule. To develop an
ICG-based NIR-fluorescent probe for SLN imaging, we synthesized
alkylated ICG molecules (ICG-Cn, n = 4, 6, 8, 10, and 18), which
are expected to be more hydrophobic than ICG (see Section 2).
Briefly, 2 types of substituted 2,3,3-trimethyl-4,5-benzo-3H-indol
were coupled using glutaconaldehyde dianil hydrogen chloride
(Scheme 2); the yields of all ICG-Cn compounds in the coupling
reaction were greater than 40%, which are acceptable as the previ-
4
5
ous report. To clarify the fluorescence properties of ICG-Cn (n = 4,
, 8, 10, and 18), we performed the absorbance and fluorescence
spectroscopy on ICG and ICG-Cn. The maximum absorbance
wavelengths of ICG-Cn in ethanol (32 M) were in the range of
87–788 nm, which is similar to that of ICG. The molar absorbance
6
l
7
5
coefficient of ICG was 1.47 Â 10 and those of ICG-Cn were in the
5
range of 1.24–2.63 Â 10 . The difference of molar absorbance
coefficients among ICG-Cn is probably due to the effect of solvent
(
ethanol) to the excitation state or molecular interaction of
ICG-Cn. However, we imply that substitution of the sulfonate
group with an alkyl chain scarcely affect the molecular energy level
of the carbocyanine chromophore. The excitation/emission spectra
of ICG-Cn in chloroform (32 lM) are shown in Figure 1. The
maximum wavelength of excitation was 766 nm for all dyes, and
the maximum wavelength of emission was in the range of
Figure 2. (a) Fluorescence excitation and emission spectra of LP-ICG-C18. (b)
Emission spectra of LP-ICG-Cn.
8
31–834 nm; these wavelength are almost identical to those of
ICG in chloroform.
ICG dissolved in aqueous solution has a maximum excitation
wavelength of 780 nm and a maximum emission wavelength of
humane conditions. A group of five rats was orally administered
LP-ICG-C18 (10 mL/kg) once a day. Another group of five rats was
intravenously administered LP-ICG-C18 (5 mL/kg) once a day. As
a reference group, five rats were administered saline, with the
same volume and frequency as both the treatment groups. The
body weights of the rats were monitored for 15 days.
41,42,55,58,59
42
8
ids,
10 nm.
In the presence of lipoproteins, phospholip-
60,74,75
60,64
or polymer colloidal particles,
the maximum excita-
tion is shifted to over 800 nm, and the maximum emission reaches
53,57,68,74,75
over 810 nm.
Since the absorbance and fluorescence
properties of ICG-Cn compounds were the same as those of ICG,
ICG-Cn compounds have potential for liposome formulation as
the NIR-fluorescent probes. Therefore, we assessed the fluores-
cence spectra of liposomally formulated ICG (LP-ICG) and ICG-Cn
3
3
. Results and discussion
.1. Fluorescence characteristics of ICG-Cn
(LP-ICG-Cn) after filtration of liposomes with a 0.4-lm pore filter.
The spectra of the fluorescence excitation/emission of LP-ICG-C18
are shown in Figure 2a. The spectra of liposomal dispersions
containing ICG-Cn (n = 4, 6, 8, and 10) is similar to that shown in
ICG, which is a water-soluble carbocyanine dye that has two
sulfonate groups (Scheme 1), has drawn much attention as a
Figure 3. (a) Absorbance spectra and (b) fluorescence emission spectra of ICG solution (blue), LP-ICG (red), and LP-ICG-C18 (black) respectively. The wavelength of excitation
is 771 nm for ICG solution and 773 nm for both LP-ICG and LP-ICG-C18. (c) Histogram of average number of photons of ICG solution (blue), LP-ICG (red), and LP-ICG-C18
(
gray) detected by the quantitative NIR-fluorescence imaging system (each NIR-fluorescence image is also shown).

T. Toyota et al. / Bioorg. Med. Chem. 22 (2014) 721–727
725
bocyanine chromophore is probably located in several different re-
gions tangential to the membrane, such as the hydrophobic region
of the condensed bushes of the alkyl chains of the lipids, the elec-
tric bilayer region comprising the polar heads of the lipids, and the
hydrophilic and polyethylene glycol-rich region above the lipo-
some surface. In contrast, the fluorescence emission spectra of
LP-ICG-Cn (n = 4, 6, 8, 10, and 18) are narrow in the range of
8
50–865 nm when they are excited at 738 nm (Fig. 2b). The peak
wavelength of the fluorescence emission exhibits a blueshift, and
the fluorescence intensity increases as the carbon number of the
substituted alkyl chain of the ICG derivatives increases. This prob-
ably results from the shift in location of the carbocyanine chromo-
phore from the vicinity of the liposome surface into the
hydrophobic region, which prevents photobleaching and redshift55
as the carbon number of the substituted alkyl chain increases. We
thus conclude that substitution of the sulfonate group of ICG with
an alkyl chain results in both an increase in the binding properties
of the carbocyanine chromophore and reinforcement of its fluores-
cence compared to ICG.
3
.2. In vitro and in vivo NIR-fluorescence imaging of LP-ICG and
LP-ICG-C18
Based on the properties of ICG-Cn and LP-ICG-Cn, we examined
Figure 4. Size distribution of LP-ICG and LP-ICG-C18. Gray bars represent the
differential frequency and the red line indicates the cumulative frequency.
NIR-fluorescence imaging with LP-ICG-C18, because the fluores-
cence intensity of LP-ICG-C18 was higher than those of the other
LP-ICG-Cn compounds (n = 4, 6, 8, and 10). When the absorbance
and fluorescence intensities of an ICG aqueous solution (glucose
isotopic solution; concentration of ICG = 0.03 mM), LP-ICG, and
LP-ICG-C18 were measured, the intensity of LP-ICG-C18 was found
to be higher than that of the ICG solution and LP-ICG (Fig. 3a and
b). By changing the concentration of ICG-C18 in the initially
Figure 2a, and it should be noted that these excitation spectra
typically exhibit a wide peak with a width of approximately
1
00 nm, whereas the emission spectra show a narrow peak. This
implies that several energy states of ICG-Cn exist when ICG-Cn
compounds are incorporated into liposomal membranes. The car-
Figure 5. (a) Time course of NIR-fluorescence images of mice in which a footpad was injected with 10 mL of LP-ICG-C18. White arrows indicate detection of the NIR-
fluorescent spot at the location of the popliteal lymph node. (b) Reference experiment data using ICG aqueous solution and LP-ICG. Not only the popliteal lymph node but also
the liver fluoresces after 30 min, and the intensity was maintained over 1 h. The enlarged views for the popliteal lymph node of each mouse were attached below the
corresponding NIR-fluorescence images.

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26
T. Toyota et al. / Bioorg. Med. Chem. 22 (2014) 721–727
prepared organic solution of the lipid mixture, we measured
LP-ICG-C18 fluorescence with a quantitative NIR-fluorescence
imaging system. We found that the fluorescence intensities of
LP-ICG-C18 and LP-ICG were statistically similar and larger than
that of the ICG aqueous solution at the concentrations of 15 and
gave brilliant fluorescence images under the NIR-fluorescence
imaging system in vitro and in vivo and remained in only the pop-
liteal lymph node in the mouse-footpad injection experiment. The
toxicity assay revealed that administration of LP-ICG-C18 via both
oral and intravenous routes had no effect on the mice. Although
further analyses are required, these results indicated that
LP-ICG-C18 would be potentially powerful for use in noninvasive
7
8
50 lM (Fig. 3c). This is probably because photobleaching of the car-
bocyanine chromophore occurs in the aggregation of the ICG solu-
tion, and ICG and ICG-C18 can merge with phospholipids in
liposomes, preventing such photobleaching. At the concentration
6
0–62,84,85
NIR-bioimaging for SLNs.
of 150
l
M, the number of fluorescence photons emitted from
Acknowledgments
LP-ICG-C18 was greater than that emitted of LP-ICG. Assuming that
the size distribution of LP-ICG and LP-ICG-C18 is the same, this is
probably because the carbocyanine chromophore of ICG is
saturated and aggregated in the liposomal membrane, whereas
ICG-C18 is likely merged in the membrane due to the substituted
alkyl chain.
We thus measured the size distribution of LP-ICG and LP-
ICG-C18 after filtration and the leak of ICG and ICG-C18 from the
liposome. Figure 4 shows the size distribution of LP-ICG-C18 and
The single-dose toxicity assay of LP-ICG-C18 was performed at
Kamakura Techno-Science Inc. (Kanagawa, Japan). This work was
partially supported by Grants-in-Aid for Scientific Research from
the Ministry of Education, Culture, Sports, Science and Technology
in Japan (No. 21790238 to A.S., No. 22501022 to Y.T., No. 23750078
to T.T. and No. 21591693 to H.H.) and a Research Grant from the
Futaba Electronics Memorial Foundation (to A.S. and Y.T.).
LP-ICG after filtration through a 0.4-
the hydrodynamic diameter of LP-ICG-C18 was 235 ± 101 nm
average ± standard deviation), which is almost same as that of
lm pore filter; we found that
Supplementary data
1
(
Supplementary data ( H NMR spectra, IR spectra, MS spectra,
LP-ICG (209 ± 79 nm). Using a preparative gel-permeable chro-
matographic column and absorbance spectrophotometer, we mea-
sured the fraction of leaked ICG or ICG-C18 separated from the
liposomal dispersion after the filtration. We found that the leaked
fraction of ICG-C18, as well as that of ICG, was less than 0.5% (see
Supplementary material). Therefore, we deduced that the influence
of ICG-C18 on liposomal properties is not different from that of ICG
and that ICG-C18 is more likely to be mono-dispersed and to fluo-
resce in the liposomal membrane than ICG at relatively high
concentrations.
and HPLC charts for each ICG-Cn, gel-filtration chromatography
charts of LP-ICG and LP-ICG-C18, and the results of single-dose tox-
icity assay using LP-ICG-C18) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.bmc.2013.12.026.
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