Preparation and characterization of phospholipid-conjugated indocyanine green as a near-infrared probe

Article abstract of DOI:10.1016/j.bmcl.2012.10.044

We have rationally designed and synthesized a novel near-infrared (NIR) photoactivating probe, designated by iDOPE, in which an indocyanine green (ICG) fluorophore is covalently conjugated with a phospholipid moiety, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), to incorporate into liposome bilayers. NIR irradiation showed that iDOPE retained the optical and fluorescence properties of ICG and demonstrated photoactivator characteristics: fluorescence emission at around 820 nm in a solvent, singlet oxygen production, and concentration-dependent heat generation. Additionally, iDOPE was incorporated into liposome bilayers and maintained stable liposomally formulated iDOPE (LP-iDOPE) over 1 week under physiological conditions. We also observed the tumor-specific biodistribution of LP-iDOPE of in vivo xenografts. These findings suggest that LP-iDOPE might be a promising tool for NIR optical imaging, photodynamic therapy, and photothermal therapy.

Full text of DOI:10.1016/j.bmcl.2012.10.044

Supporting Information
Preparation and characterization of phospholipid-conjugated
indocyanine green as a near-infrared probe
Akiko Suganamia, Taro Toyotaa,b, Shigetoshi Okazakic, Kengo Saitod, Katsuhiko
Miyamotoe, Yasunori Akutsuf, Hiroshi Kawahirag, Akira Aokih, Yutaka Murakih,
Tomoyuki Madonoh, Hideki Hayashig, Hisahiro Matsubaraf, Takashige Omatsue,
Hiroshi Shirasawad, Yutaka Tamuraa,*
a Department of Bioinformatics, Chiba University
b The University of Tokyo
c Hamamatsu University School of Medicine
d Department of Molecular Virology, Chiba University
e
Department of Image and Material Science, Chiba University
f Department of Frontier Surgery, Chiba University
g Research Center for Frontier Medical Engineering, Chiba University
h Yamada Chemical Co., Ltd.
S1

Table of Contents
Materials and Methods ……………………………………….. S3
Molecular Modeling …………………………………………. S3
Syntheses …………………………………………………...
S3
NMR Measurements …………………………………………. S6
FTIR Measurements …………………………………………. S6
HPLC Analysis ………………………………………………. S6
UV-vis Fluorescence Spectroscopy ………………………….. S6
Singlet Oxygen Measurements ………………………………. S7
Measurement of Photothermal Properties …………………… S7
Preparation of Liposomes …………………………….……… S8
Gel-filtration Chromatography ………………………………
S9
Measurement of Particle Size ………………………………... S9
Biodistribution of LP-iDOPE by Liposome Formulas……….. S9
Results ………………………………………………………….. S10
Molecular Modeling: Figure S1……………………………… S10
NMR Measurements: Figure S2 ……………………………... S11
NMR Measurements: Figure S3 ……………………………... S12
FTIR Measurements: Figure S4.……………………………... S13
FTIR Measurements: Table S1 ………………………………. S13
FTIR Measurements: Figure S5.……………………………... S14
FTIR Measurements: Table S2.………………………………. S14
HPLC Analysis Figure: S6 …………………………………... S15
Singlet Oxygen Measurements: Figure S7 …………………... S16
Dynamic Light-scattering Measurements: Figure S8.………... S17
Gel-filtration Chromatograph: Figure S9…………………….. S18
S2

Materials and Methods
Molecular Modeling
The three-dimensional structure of ICG was constructed using MOE (version 2009,
CCG Inc., Montreal, Canada) according to PubChem Compound CID 11967809
(National Center for Biotechnology Information, Bethesda MD, USA). The
three-dimensional
structure
of
DOPE
was
constructed
using
VMD
(http://www.ks.uiuc.edu/). Then, the three-dimensional structures of ICG and DOPE
were used to construct that of iDOPE using the build command in MOE. Molecular
mechanics calculations were performed to obtain the local minimum structures of ICG,
DOPC, and iDOPE, using the AMBER99 force field in MOE. Then, the lipophilic
colors of the surfaces of ICG, DOPC, and iDOPE were calculated from the Wildman
and Crippen SlogP parameter in MOE.
Syntheses
iDOPE was prepared as shown in Scheme 1. The ICG fluorophore was synthesized
from a single starting material, 2,3,3-trimetyl-4,5-benzo-3H-indole (1). 1 (3.1 g, 15
mmol) was added to acetonitrile (10 mL) with 3-bromo-1-propionic acid (1.5 g, 9.8
mmol), and the reaction solution was stirred at 65 °C for 16 h and then cooled to room
temperature. Ethyl acetate (50 mL) was then added to the solution and the product,
2,3,3-trimetyl-1-(2-carboxyethyl)-4,5-benzoindolium bromide (2), was separated by
filtration and rinsed with acetone, giving 1 as gray crystals (1.68 g, 47%). Then 1 (3.1 g,
15 mmol) and 1,4-butane sultone (2.1 g, 15 mmol) were mixed in a round-bottomed
flask (25 mL) under a nitrogen atmosphere, and the reaction mixture was stirred at 80
°C for 4 h and then cooled to room temperature. On addition of acetone to the reaction
S3

mixture the residue dissolved, and the product was crystallized. The reaction product,
2,3,3-trimethyl-1-(sulfobutyl)-4,5-benzoindolium inner salt (3), was separated by
filtration and rinsed with acetone to give 3 as gray crystals (1.17 g, 23%). A mixture of
3 (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 to room temperature to crystallize
the
product,
2-(6-acetoanilido-1,3,5-hexatrienyl)-3,3-dimethyl-1-(sulfobutyl)-4,5-benzo[e]indolium
inner salt (4). This salt was separated by filtration and rinsed with acetone to give 4 as
dark-purple crystals (0.97 g, 58%). A pyridine solution (8 mL) of 2 (0.55 g, 1.5 mmol)
and 4 (0.80 g, 1.5 mmol) was stirred at 50 °C for 45 min, and then cooled to room
temperature. The reaction mixture was condensed under reduced pressure, and then
distilled water (20 mL) and 10% HCl were added to adjust the pH to 3–4. The crude
product was filtered from the reaction mixture and dried. After crystallization using
methanol/chloroform (volume ratio: 5/1, 20 mL) and recrystallization in
methanol/chloroform,
the
product,
4-(2-((1E,3E,5E,7E)-7-(3-(2-carboxyethyl)-1,1-dimethyl-1H-benzo[e]indol-2(3H)-ylide
ne)hepta-1,3,5-trienyl)-1,1-dimethyl-1H-benzo[e]indolium-3-yl)butane-1-sulfonic acid
(5), was obtained as dark-green crystals (0.38 g, 37%). In a mixed organic solvent of
acetonitrile (3 mL) and chloroform (10 mL), 5 (0.5 g, 0.73 mmol) and
N-hydroxysuccinimide (0.17 g, 1.5 mmol) were dissolved, and a chloroform solution (2
mL) of N,Nʹ-dicyclohexylcarbodiimide (0.30 g, 1.5 mmol) was added dropwise at 2 °C
under a nitrogen atmosphere. The reaction solution was stirred at room temperature for
30 min. The residue was separated by filtration and the reaction solution was condensed
S4

under reduced pressure, with the temperature kept below 25 °C. The crude product was
rinsed with ethyl acetate and acetone.
The crude product (0.66 g) was dissolved in chloroform (10 mL) in a round-bottomed
flask
(25
mL)
and
a
chloroform
solution
(5
mL)
of
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (0.54 g, 0.73 mmol) and triethylamine
(0.10 g, 1.0 mmol) was added dropwise to the reaction solution over 20 min. The
reaction mixture was stirred at room temperature for 2.5 h and the residue was separated
by filtration. The obtained solution was condensed and purified using silica column
chromatography with methanol/chloroform (volume ratio: 1/4-1/3). The product 6 was
obtained as a green powder (0.37 g, 36%).
5: 1H NMR (DMSO-d6): δ (ppm) 1.90 (s, 14H); 2.54 (t, 2H); 2.75 (t, 2H); 4.25 (br, 2H);
4.38 (br, 2H); 6.34 (d, 1H); 6.52~6.68 (m, 3H); 7.44~7.59 (m, 6H); 7.76~7.97 (m, 3H);
8.01~8.10 (m, 6H); 8.20~8.29 (m, 2H); 12.6 (br, 1H).
6: 1H NMR (DMSO-d6): δ (ppm) 0.83 (t, 6H), 1.15–1.22 (m, 52H), 1.48 (t, 4H), 1.82 (q,
4H), 2.22 (t, 4H), 2.55 (m, 4H), 3.03 (q, 6H), 3.16 (t, 4H), 3.66 (quintet, 2H), 3.75 (t,
2H), 4.07 (m, 1H), 4.25 (t, 3H), 4.41 (t, 2H), 5.07 (m, 1H), 5.29 (m, 4H), 6.36 (d, 1H),
6.52–6.62 (m, 3H), 7.47 (m, 2H), 7.61–7.66 (m, 3H), 7.65 (m, 2H), 7.90–8.07 (m, 6H),
8.22 (d, 2H), 8.73 (t, 1H), 10.0 (br, 1H).
S5

NMR Measurements
The 1H NMR spectra of iDOPE (6 in Scheme 1) and intermediate (5 in Scheme 1) were
run at 300 MHz in DMSO-d6 using a Lambda-300 NMR spectrometer (JEOL, Tokyo,
Japan).
FTIR Measurements
Infrared spectra of iDOPE (6 in Scheme 1) and intermediate (5 in Scheme 1) were
recorded as potassium bromide pellets using an IRPrestige-21 spectrometer (Shimadzu,
Kyoto, Japan) in the 4000–400 cm−1 region.
HPLC Analysis
iDOPE was analyzed by HPLC using a Shimadzu LC-20A chromatography system
(Shimadzu, Kyoto, Japan) equipped with CHEMCOSORB 7-ODS-L (7 µm, 4.6 mm ×
150 mm, Chemco Scientific, Osaka, Japan). The column temperature was controlled at
45 °C using a CTO column oven (Shimadzu).
Eluent: [0.02M KH2PO4 (pH3.0 with H3PO4)/CH3OH (95/5)]/CH3OH/THF: 21/50/29.
Flow rate: 1.2 mL/min.
UV-vis-NIR and Fluorescence Spectroscopy
The absorption spectra of iDOPE and ICG dissolved in organic solvents were obtained
using a UV-vis-NIR spectrometer (UV-3500, Shimadzu, Kyoto, Japan).
Fluorescence emissions from iDOPE and ICG were observed using a fluorescence
spectrometer (F-4500, Hitachi, Tokyo, Japan).
S6

Singlet Oxygen Measurements
The luminescence from singlet oxygen was measured using a high-sensitivity detection
method (NIR-PII System, Hamamatsu Photonics, Hamamatsu, Japan). The excitation
pulse was obtained using an optical parametric oscillator (L5996, Hamamatsu
Photonics), which was excited using an Nd:YAG laser (Surelite I-20, Continuum, Santa
Clara, CA, USA). The pulse width and intensity were approximately 5 ns and 300
µJ/pulse, respectively, and the repetition rate was 20 Hz. The singlet oxygen
luminescence was monitored using an IR-gated image intensifier (NIR-PII, Hamamatsu
Photonics) after passage through a polychromator (MS257, Oriel Instruments, Stratford,
CT, USA). We began measurements at 5 µs after excitation, and the exposure time was
100 µs. The signals were accumulated by repeated detection (2000 times). The
calibration of the wavelength was performed using a spectral calibration lamp (Krypton
type, Oriel Instruments, Stratford, CT, USA).
Measurement of Photothermal Properties
iDOPE and ICG in a mixed organic solvent of CH3OH/CHCl3 (volume ratio: 1/9, 1.5
mL) in
a
quartz cuvette were irradiated using an 808-nm laser
(LU0808T020‐E105N12A, Hanamura Optics, Yokohama, Japan) at 0.5 W/cm2. The
solution temperature was measured at 1-s intervals using an SF Series Temperature
Probe (AM-8000, Anritsu Meter, Tokyo, Japan) under stirring.
S7

Preparation of Liposomes
1. LP-iDOPE
Cholesterol (1.0 mM, Nippon Fine Chemical Co., Ltd., Tokyo, Japan),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, 10 mM, NOF Corporation, Tokyo,
Japan),
N-(carbonyl-methoxypolyethyleneglycol
5000)-1,2-distearoly-sn-glycero-3-phosphoethanolamine sodium salt (DSPE-PEG, 0.5
mM, NOF Corporation), and iDOPE (5.0 × 10−5, 1.0 × 10−4, 5.0 × 10−4, and 1.0 × 10−3
mM, iDOPE/DOPC molar ratio of 0.005, 0.01, 0.05, and 0.1) were dissolved in a mixed
organic solvent of CH3OH/CHCl3 (volume ratio: 1/9). A thin lipid film was formed by
removal of the solvent under reduced pressure. After addition of an aqueous buffered
solution (phosphate buffered saline [PBS], pH = 7.4) at room temperature, the liposome
dispersion was filtered through a 0.1-mm pore polycarbonate filter attached to a
LiposoFast-Stabilizer (Avestin Inc., Ottawa, Canada).
2. LP-ICG
Cholesterol (1.0 mM, NOF Corporation), DOPC (10 mM, NOF Corporation), and
DSPE-PEG (0.5 mM, NOF Corporation) were dissolved in a mixed organic solvent of
CH3OH/CHCl3 (volume ratio: 1/9). A thin lipid film was formed by removal of the
solvent under reduced pressure. The dried thin lipid film, consisting of cholesterol,
DOPC, and DSPE-PEG, was hydrated with PBS containing ICG (1.0 × 10−4, 2.5 × 10−4,
and 5.0 × 10−4 mM, ICG/DOPC molar ratio of 0.01, 0.025, and 0.1) and the liposome
dispersion was filtered through a 0.1-mm pore polycarbonate filter attached to a
LiposoFast-Stabilizer (Avestin Inc.)
S8

Gel-filtration Chromatography
LP-iDOPE and LP-ICG were separated from free iDOPE and ICG, respectively, using
PD-10 Desalting Columns containing Sephadex G-25 (GE Healthcare Bio-Sciences AB,
Uppsala, Sweden). The absorbance of the obtained fraction (1 mL) was measured using
a UV-vis-NIR spectrometer (UV-1800, Shimadzu, Kyoto, Japan).
Measurement of Particle Size
Particle size analysis of LP-iDOPE was performed by dynamic light-scattering
measurements (SZ-100, HORIBA Ltd., Kyoto, Japan).
Biodistribution of iDOPE by Liposome Formulas
Female BALB/c Slc (nu/nu) mice that were 5-6 weeks old were purchased from the
Japan SLC, Inc. (Hamamatsu, Japan). Tumors were initially established by
subcutaneously injecting a mixture of 1 × 10−7 A549 cells (adenocarcinomic human
alveolar basal epithelial cells), matrigel, and DMEM. Tumor volume was calculated
π/6ab2, where a is the length and b is the width of the tumor. Mice received an
intravenous injection of LP-iDOPE, when the tumors reached a volume of 170 mm3.
The biodistribution of LP-iDOPE was studied by injecting LP-iDOPE, containing 14.0
mg/kg of iDOPE, intravenously through a tail vein of mice bearing A549 tumors and
imaged 24, 48, and 144 h after the injection with an In-Vivo MS FX PRO imaging
system (Carestream Health, New York, USA)
S9

Results
Molecular Modeling
Figure S1. Predicted three-dimensional structures of (a) ICG, (b) DOPC, and (c)
iDOPE. Blue: hydrophilic region; yellow: hydrophobic region.
S10

NMR Measurements
Figure S2. 1H-NMR spectrum of iDOPE.
S11

Figure S3. 1H-NMR spectrum of intermediate 5 in Scheme 1.
S12

FTIR Measurements
Figure S4. FTIR spectrum of iDOPE.
Table S1
Observed assignments of iDOPE
Experimental Frequency (cm-1)
Vibrational Assignments
2924.09
2852.72
1753.93
1654.92
1527.62
1508.33
1089.78
νCH
νCH
νC=O
νC=C, γCN +γNH + νC=O
νC=C
νC=C
νS-O
ν: stretching, γ: out-of-plane bending
S13

Figure S5. FTIR spectrum of intermediate 5 in Scheme 1.
Table S2
Observed assignments of intermediate 5 in Scheme 1
Experimental Frequency (cm-1)
Vibrational Assignments
2976.16
2933.73
1718.58
1508.33
1421.54
1354.03
1089.78
923.90
νCH
νCH
νC=O
νC=C
νC=C
νS(=O)2
νS-O
γCH
665.44
γCC
ν: stretching, γ: out-of-plane bending
S14

HPLC Analysis
Figure S6. HPLC chromatograms of iDOPE: (a) 254 nm and (b) 780 nm.
S15

Singlet Oxygen Measurements
Figure S7. Luminescence spectrum of singlet oxygen in oxygen-saturated CHCl3
solution of iDOPE (32 µM, solid line). The dotted line represents the luminescence
spectrum of iDOPE in the presence of β-carotene (6 µM).
The excitation wavelength was 690 nm. The pulse width and intensity were
approximately 10 ns and 300 µJ/pulse, respectively, and the repetition rate was 30 Hz.
Measurements were begun at 30 µs after excitation, and the exposure time was 200 µs.
The signals were accumulated by repeated detection (1500 times).
S16

Dynamic Light-scattering Measurements
Figure S8. Diameter distribution of LP-iDOPE. iDOPE/DOPC molar ratios are 0 (blue
line), 0.005 (red line), 0.01 (green line), 0.05.(black line), and 0.1 (magenta line).
S17

Gel-filtration Chromatograph
Figure S9. Gel-filtration chromatograph of (a) LP-ICG (ICG/DOPC molar ratios 0.01
and 0.1 mM of ICG) and (b) ICG (0.1 mM) on PD-10 Desalting Columns containing
Sephadex G-25. Photographs of gel-filtration column were taken during the fraction
tube exchange at every 1 ml elusion.
S18