Design and synthesis of fluorescence-labeled closo-dodecaborate lipid: Its liposome formation and in vivo imaging targeting of tumors for boron neutron capture therapy

Article abstract of DOI:10.1039/c1ob06500a

The fluorescence-labeled closo-dodecaborane lipid (FL-SBL) was synthesized from (S)-(+)-1,2-isopropylideneglycerol as a chiral starting material. FL-SBL was readily accumulated into the PEGylated DSPC liposomes prepared from DSPC, CH, and DSPE-PEG-OMe by the post insertion protocol. The boron concentrations and the fluorescent intensities of the FL-SBL-labeled DSPC liposomes increased with the increase of the additive FL-SBL, and the maximum emission wavelength of the liposomes appeared at 531 nm. A preliminary in vivo imaging study of tumor-bearing mice revealed that the FL-SBL-labeled DSPC liposomes were delivered to the tumor tissue but not distributed to hypoxic regions.

Full text of DOI:10.1039/c1ob06500a

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PAPER
Design and synthesis of fluorescence-labeled closo-dodecaborate lipid: its
liposome formation and in vivo imaging targeting of tumors for boron neutron
capture therapy†
Hiroyuki Nakamura,* Noriko Ueda, Hyun Seung Ban, Manabu Ueno and Shoji Tachikawa
Received 1st September 2011, Accepted 1st November 2011
DOI: 10.1039/c1ob06500a
The fluorescence-labeled closo-dodecaborane lipid (FL-SBL) was synthesized from
(S)-(+)-1,2-isopropylideneglycerol as a chiral starting material. FL-SBL was readily accumulated into
the PEGylated DSPC liposomes prepared from DSPC, CH, and DSPE-PEG-OMe by the post
insertion protocol. The boron concentrations and the fluorescent intensities of the FL-SBL-labeled
DSPC liposomes increased with the increase of the additive FL-SBL, and the maximum emission
wavelength of the liposomes appeared at 531 nm. A preliminary in vivo imaging study of tumor-bearing
mice revealed that the FL-SBL-labeled DSPC liposomes were delivered to the tumor tissue but not
distributed to hypoxic regions.
the targeting of boron to tumors.14 In particular, liposomes
Introduction
are a promising approach as they can carry relatively large
Since the discovery of the enhanced permeability and reten-
quantities of boron compounds with selective localization in
tion (EPR) effect by Maeda and Matsumura in 1986,1,2 much
attention has been focused on drug delivery systems (DDS)
using drug-embedded nano carriers including liposomes, micelles,
proteinaceous or polymer-conjugated macromolecules, and lipid
particles.3,4 The EPR effect is based on the abnormal architectures
and impaired functional regulation of newly formed tumoral
blood vessels, and this phenomena will lead to abnormal molecular
and fluid transport dynamics especially for macromolecular
species.5,6
This nano carrier-based DDS is also an attractive approach
for efficient boron delivery systems (BDS) to tumors for boron
neutron capture therapy (BNCT). BNCT is a bimodal therapy
reliant on the selective uptake and/or retention of sensitizing
boron molecules by tumor cells and activation of those sensitizers
by thermal neutrons as an external radiation source. The neutron
capture reaction with the boron-10 nucleus yields high linear
energy transfer (LET) particles, 4H and 7Li, with a range of 5–9 mm,
thus BNCT has been expected as a cell-selective radiation therapy
with the potential to control local recurrences of malignant
tumors. For successful therapy, boron concentrations in the
tumor of around 20–30 ppm are required with sufficiently low
toxicity.7–13 Therefore, development of efficient boron delivery
to tumors is still am important requirement in BNCT. Various
nano carrier-based BDS approaches have been employed for
tumors.15–18
We have focused on the structures of phospohrous lipids in the
liposomal bilayer and first developed the boron cluster lipid.19
Although the liposomes prepared from the nido-carborane lipid
accumulated in the tumor,20 their acute toxicity became a serious
problem at 14 mg boron per kg body weight in mice.20,21 We
succeeded in the replacement of the nido-carborane by closo-
dodecaborate in the boron cluster lipids.22,23 The liposomes pre-
pared the closo-dodecaborate lipids did not display acute toxicity
for up to 30 mg boron per kg body weight in mice24 and a boron
concentration of 23 ppm in the tumor with a tumor/blood ratio of
~2 was observed 24 h after administration of the distearoylphos-
phathidyl boron lipid (DSBL)-25% PEG liposomes.25 Symmetric
closo-dodecaborate lipids and monoanionic closo-dodecaborate
lipids have been recently developed as candidates for liposomal
boron delivery vehicles.26–28 Therefore, a boron delivery system
(BDS) to tumors using boron-lipid liposomes has become one
of the most promising approaches for BNCT. In BDS, it is
very important to know detailed boron biodistribution in tissues
because the distributions of tumor cells are often heterogeneous
and liposomes may spread over a certain distance from tumoral
blood vessels. Although the boron concentration in each tissue
has been determined using inductively coupled plasma (ICP)
or g-prompradiography, a cell-level distribution of boron lipid
liposomes has not been studied yet. In this paper, we report the
design and synthesis of fluorescence-labeled closo-dodecaborane
lipids (FL-SBL) (see Fig. 1) and the preparation of FL-SBL-
labeled DSPC liposomes. We preliminarily demonstrated an in
vivo biodistribution experiment of the FL-SBL-labeled DSPC
liposomes using tumor-bearing mice.
Department of Chemistry, Faculty of Science, Gakushuin University, 1-5-1
Mejiro, Toshima-ku, Tokyo, 171-8588, Japan. E-mail: hiroyuki.nakamura@
gakushuin.ac.jp; Fax: (+81) (0)3 5992 1029; Tel: (+81) (0)3 3986 0221
† Electronic supplementary information (ESI) available: spectral data for
new compounds. See DOI: 10.1039/c1ob06500a
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Fig.
1
Structures of distearoylphosphatidylcholine (DSPC), closo-
dodecabarate lipid (DSBL), and fluorescence-labeled closo-dodecabarate
lipid (FL-SBL).
Scheme 2 Reagents and conditions: (a) 12-aminododecanoic acid,
NaHCO3, EtOH, 37 ◦C, 56%; (b) 3, DCC, DMAP,CHCl3, 55%; (c)
HF-pyridine, pyridine, THF; (d) bromoacetylbromide, pyridine, CHCl3,
68% in two steps.
Results and discussion
Synthesis
Synthesis of the hydrophobic tail function of FL-SBL is
shown in Scheme 1. The coupling reaction of (S)-(+)-
1,2-isopropylideneglycerol (1) with stearic acid proceeded in
the presence of dicyclohexylcarbodiimide (DCC) and N,N-
dimethylaminoprydine (DMAP) in CHCl3 to give the correspond-
ing ester, which was treated with Amberlyst 15 in MeOH/CH2Cl2
(1 : 1) to afford the diol 2 in 88% yield in two steps.29 Selective
protection of the primary alcohol in the diol 2 with TBSCl gave
the corresponding silyl ether 3 in 85% yield.
Scheme 3 Reagents and conditions: (a) i. 7, CH3CN, ii. chromatography
on SiO2, 23%; (b) t-BuOK, THF, 49%.
(1 : 1) and the resulting S-dialkylated dodecaborate was treated
with tetramethylammonium hydroxide in dry THF to give the
protected BSH 8. S-Alkylation of 8 with the precursor 7 proceeded
in acetonitrile under reflux gave the corresponding S-dialkylated
product 9, which was treated with potassium tert-butoxide in dry
THF to afford the fluorescence-labeled closo-dodecaborane lipid
FL-SBL.
Scheme 1 Reagents and conditions: (a) octadecanoic acid, DMAP, DCC,
CHCl3, 88%; (b) Amberlyst 15, MeOH/CH2Cl2, quant; (c) TBDMSCl,
imidazole, THF, 85%.
We next synthesized the fluorescent tail of the boron lipid.
We chose 4-amino-7-nitrobenzofurazan (NBF) as a fluorescent
moiety and introduced it into one of two tails in DSBL, as shown
in Scheme 2. The reaction of chloro-7-nitrobenzofurazan with
12-aminodecanoic acid proceeded in the presence of NaHCO3 in
ethanol to afford the corresponding NBF-conjugated decanoic
acid 4 in 56% yield.30 The coupling reaction of 329,31 with 4 gave
the fluorescence-labeled hydrophobic tail function 5 in 55% yield.
Removal of the TBS group in compound 5, followed by ester
formation with bromoacetylbromide afforded the fluorescence-
labeled lipid precursor 6 in 68% yield in two steps.
Characterization of FL-SBL liposomes
PEG units conjugated on the surface of liposomes are important
for a prolonged residence time in the circulation and escaping
ability from RES uptake, promoting the extravasation of the
liposomes into the solid tumor tissue for the liposomal drug
delivery system. Therefore, we prepared PEG-conjugated FL-
SBL-labeled DSPC liposomes by means of post-insertion of
various amounts of FL-SBL into PEGylated DSPC liposomes.
The boron and phosphorus concentration in the liposome so-
lutions and their diameter and zeta potential were measured.
The results are summarized in Table 1. The PEGylated DSPC
Scheme 3 shows the conjugation of a closo-dodecaborate moiety
to the precursor 7 using the protected BSH 8, which was prepared
from BSH according to Gabel’s protocol. Briefly, BSH was
reacted with 2-bromopropionitrile (2 equiv.) in CH3CN/H2O
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Table 1 Boron and phosphorus concentrations, particle size, and zeta potential of the FL-SBL-labeled PEGylated DSPC liposomes
Volume of FL-SBL (mL)
Boron conc. (ppm)
Phosphorus conc. (ppm)
B/P ratio (%)
Particle size (nm)
Zeta potential (mV)
0
10
20
30
40
—
1876.2
2153.4
2169.2
2066.1
2054.5
0
97.2 0.15
97.6 0.47
97.4 0.20
96.2 1.38
97.8 1.15
4.10 0.35
23.2 2.35
23.3 3.04
21.1 1.70
20.0 3.52
41.3
73.8
112.6
146.7
1.9
3.4
5.4
7.1
liposomes prepared from DSPC, CH, and DSPE-PEG-OMe
(1 : 1 : 0.11, molar ratio) by REV method displayed 97.22 nm in
diameter with -29.3 mV of zeta potential. Then various volumes
(10–40 mL) of the FL-SBL solution (10 mg mL-1) were mixed
with 500 mL of the primary prepared PEGylated DSPC liposome
solution (1876 ppm P concentration). Free FL-SBL was removed
from the resulting liposome solutions by ultracentrifugation at
200,000 g for 60 min. With the increase in volume of the additive
FL-SBL solution, the boron concentration in the liposomes
increased. The boron/phosphorus concentration ratio increased
roughly in proportion from 1.9 to 7.1% w/w without affecting their
particle size. However, addition of the FL-SBL solution increased
the zeta potential of each liposome from -29.3 to -20.0 mV. Our
previous results showed that the zeta potential of the 5% DSBL
liposomes is -42.8 mV and an increase in the anionic DSBL lipid
contents resulted in more anionic liposome formation.25 Since the
current liposomes contain the PEG function, the aqueous surface
layers of liposomes formed by the PEG function would involve
the counter cations of negatively charged FL-SBL. Thus, the
PEGylated DSPC liposomes became more cationic by addition
of FL-SBL.
We next investigated a concentration-dependent increase in
fluorescence intensity of the FL-SBL-labeled DSPC liposomes.
The fluorescence intensity was measured at a range of emission
wavelengths from 475 to 650 nm using an excitation wavelength of
460 nm. As shown in Fig. 2, the maximum emission wavelength
of FL-SBL-labeled DSPC liposomes appeared at 531 nm and
the fluorescent intensities increased along with increase of the
boron/phosphorus concentration ratios that are indicated in
Table 1. A slight shift in the wavelength of maximum fluorescence
emission toward higher wavelength was observed along with
increase of a concentration of FL-SBL in the liposome membrane.
In vivo imaging
We next examined the in vivo biodistribution of the FL-SBL-
labeled liposomes using the colon 26 tumor-bearing mice. The
tumor-bearing mice were injected via the tail vein with 200 mL of
FL-SBL-labeled liposomes. After 36 h, the tumor was excised and
frozen in O.C.T. embedding compound, and a cryostat section
was prepared for immunofluorescent staining. Fig. 3 shows the
fluorescence microscopy images of cryostat sections of tumor
tissues injected with the FL-SBL-labeled liposomes (green) and
cell nuclei were stained with DAPI (blue). Hypoxic cells were
visualized by immunofluorescent staining with an endogenous
marker of tumor hypoxia, anti-HIF-1a antibody (red). Interest-
ingly, the distribution of the FL-SBL-labeled liposomes did not
overlap with the localization of hypoxic cells. In general, hypoxic
cells are located a certain distance from blood vessels (>150 mm)
because of the low oxygen supply. Therefore, the current results
may indicate that the liposomes are delivered to the tumor tissue
but not distributed to hypoxic tissue which is located far from the
blood supply in the tumor. Previously we reported the BNCT
effect of tumor-bearing mice injected with dodecaborate lipid
(DSBL) liposomes.25 Tumor growth inhibition was observed a
week after neutron irradiation, however, re-growth of the tumor
Fig. 3 In vivo imaging of the FL-SBL-labeled liposomes in the colon 26
tumor-bearing mice. The tumor-bearing mice were injected via the tail vein
with 200 mL of FL-SBL-labeled liposomes. After 36 h, the tumor tissues
were stained with DAPI (blue). Immunofluorescent staining (red) was
carried out with an endogenous marker of tumor hypoxia, anti-HIF-1a
antibody. Distribution of the FL-SBL-labeled liposomes in tumor tissue
was visualized by green.
Fig. 2 Dose-dependent increase of fluorescence intensity of the FL-S-
BL-labeled PEGylated liposomes.
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was also observed after two weeks. It is known that HIF-1a is a key
regulator of the expression of various genes associated with tumor
angiogenesis, metastasis, invasion, proliferation and apoptosis.32
HIF-1a is implicated in treatment resistance and poor prognosis in
the hypoxic region around the cancer. Therefore, delivery of drugs
into tissues located far from blood vessels has been considered as
an important issue for successful drug targeting in drug delivery
systems.33 In this regard, delivery of boron to the hypoxic regin is
also an important issue for the achievement of efficient BNCT of
cancers.
(R)-(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl stearate
To a solution of (S)-(+)-1,2-isopropylideneglycerol (1) (2.00 g,
15.1 mmol) in chloroform (20 mL) were added stearic acid
(4.85 g, 17.1 mmol), 4-dimethylaminopyridine (DMAP: 2.09 g,
17.1 mmol) and the mixture was stirred for 25 min. To this reaction
mixture was added dicyclohexylcarbodiimide (DCC: 3.53 g,
17.1 mmol) in chloroform (12 mL) dropwise and the mixture
was further stirred for 2 h at room temperature. The mixture
was filtered with celite and the filtrate was concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography with hexane/ethyl acetate (50 : 1) to give (R)-
(2,2-dimethyl-1,3-dioxolan-4-yl)methyl stearate as a white solid
Conclusion
1
(6.00 g, 1.51 mmol, 88%): H NMR (400 MHz; CDCl3) d 4.35–
We succeeded in the synthesis of the fluorescence-labeled closo-
dodecaborane lipid, FL-SBL, which was readily accumulated
into the PEGylated DSPC liposomes prepared from DSPC, CH,
and DSPE-PEG-OMe by the post insertion protocol. The boron
concentrations and the fluorescence intensities of the resulting
FL-SBL-labeled DSPC liposomes increased with an increase of
the additive FL-SBL and the maximum emission wavelength
of the liposomes appeared at 531 nm. A preliminary in vivo
imaging study of the boron liposomes using colon 26 tumor-
bearing mice showed that the boron liposomes are delivered to
the tumor tissue but not distributed to hypoxic tissue which is
located far from the blood supply in the tumor. In our previous
study, we found that boron concentrations in various organs
became undetectably low within three weeks after injection.25
Thus, we can demonstrate the stability of the boron lipids under
biological conditions after accumulation in organs using FL-SBL.
If the dodecaborate moiety and fatty acid moieties of FL-SBL
are readily cleaved under biological conditions, the fluorescent-
conjugated fatty acids are accumulated in tissues and detectable by
fluorescent microscopic analysis although boron concentration is
low. Although more detailed biodistribution studies are necessary
to clarify the behavior of boron liposomes in vivo, the current
investigation involves an important aspect for further development
of liposomal boron delivery systems in BNCT.
4.29 (m, 1H), 4.16 (dd, J = 11.2, 4.4 Hz, 1H), 4.08 (ddd, J = 8.8,
6.0 Hz, 2H), 3.74 (dd, J = 7.4 Hz, 1H), 2.34 (t, J = 7.6 Hz, 2H),
1.64–1.61 (m, 2H), 1.44 (s, 3H), 1.37(s, 3H), 1.28–1.25 (m, 28H),
0.88 (t, J = 6.4 Hz, 3H); 13C NMR (75 MHz, CDCl3) d 173.7,
109.8, 73.7, 66.3, 64.5, 34.1, 31.9, 29.7, 29.4, 29.3, 26.7, 25.4, 24.9,
22.7, 14.1; IR (KBr) 2920, 2851, 1736 cm-1; MS (ESI) m/z 421
([M+Na]+); Anal. Calcd for C24H46O4: C, 72.31; H, 11.63. Found:
C, 72.06; H, 11.55.
(R)-2,3-Dihydroxypropyl stearate (2)
(R)-(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl stearate (7.00 mg,
17.6 mnol) was dissolved in methanol (60 mL) and
dichloromethane (30 mL) and Amberlyst 15 (11.7 g) was added.
The mixture was stirred under reflux for 2 h and then the resign
was removed by filtration. The solvents were evaporated under
reduced pressure and the residue was purified by silica gel column
chromatography with hexane/ethyl acetate (5 : 1) to give (R)-2,3-
dihydroxypropyl stearate (2) as a white solid (6.31 g, 17.6 mmol,
quant): 1H NMR (400 MHz; CDCl3) d 4.24–4.13 (m, 1H), 3.99–
3.92 (m, 1H), 3.73–3.58 (m, 3H), 2.37–2.28 (m, 2H), 1.63–1.61
(m, 2H), 1.28–1.26 (m, 28H), 0.88 (t, J = 6.8 Hz, 3H); 13C NMR
(75 MHz, CDCl3) d 174.3, 70.2, 65.1, 63.3, 34.1, 31.9, 29.6, 29.4,
29.1, 24.8, 22.6, 14.0; IR (KBr) 2920, 2851, 1732, 1180 cm-1;
MS (ESI) m/z 359 ([M+H]+), 381 ([M+Na]+); Anal. Calcd for
C21H42O4: C, 70.34; H, 11.81. Found: C, 70.46; H, 11.86.
Experimental section
1H NMR and 13C NMR spectra were measured on JEOL
JNM-AL 300 (300 MHz) and VARIAN UNITY-INOVA 400
(400 MHz) spectrometers. Chemical shifts in the 1H and 13C NMR
spectra were expressed in parts per million (ppm, d units) and
coupling constant was expressed in units of hertz (Hz). IR spectra
were measured on a Shimadzu FTIR-8200A spectrometer. High-
resolution mass spectra (ESI) were recorded on a Bruker Daltonics
micro TOF-15 focus. Elemental analyses were performed by a CE
instrument EA1110 CHNS-O automatic elemental analyzer. Ana-
lytical thin-layer chromatography (TLC) was performed on a glass
plates (Merck Kieselgel 60 F254, layer thickness 0.2 mm). Column
chromatography was performed on silica gel (Merck Kieselgel 70–
230 mesh). All reactions were carried out under argon atmosphere
using standard Schlenk techniques. Most chemicals and solvents
were of analytical grade and used without further purification.
Distearoylphosphatidylcholine (DSPC) (COATSOME MC-8080)
and DSPE-PEG-OMe (DSPE-020C) were supplied by Nippon Oil
and Fats (Tokyo, Japan).
3-O-Stearoyl-1-O-(tert-butyl-dimethyl-silanyl)-sn-glycerol (3)
To a mixture of 2 (3.22 g, 8.98 mmol) and imidazole (0.88 g,
12.5 mmol) in THF (50 mL) was added a THF (25 mL) solution
of tert-butyldimethylsilyl chloride (1.51 g, 9.99 mmol) dropwise
and the mixture was stirred for 2 h under reflux. The solvent
was removed under reduced pressure and the residue was purified
by silica gel column chromatography with hexane/ethyl acetate
(20 : 1) to give 3-O-stearoyl-1-O-(tert-butyl-dimethyl-silanyl)-sn-
1
glycerol (3) as a white solid (3.60 g, 7.61 mmol, 85%): H NMR
(400 MHz; CDCl3) d 0.08 (s, 6H), 0.88 (t, J = 6.8 Hz, 3H), 0.90 (s,
9H), 1.25 (s, 28H), 1.60–1.64 (m, 2H), 2.34 (t, J = 7.6, 2H), 3.67
(dd, J = 9.4 Hz, J = 18.8 Hz, 2H), 3.86–3.89 (m, 1H) 4.09–4.18 (m,
2H); 13C NMR (300 MHz; CDCl3) d -5.47, 14.10, 18.26, 22.68,
24.94, 25.82, 29.14, 29.26, 29.35, 29.45, 29.68, 31.92, 34.20, 63.70,
64.98, 70.01, 173.95; IR (CHCl3) 2928, 2856, 1734, 1464, 1258,
1177, 1096 cm-1; MS (ESI) m/z 496 ([M+Na]+); Anal. Calcd for
C27H56O4Si: C, 68.59; H, 11.94. Found: C, 68.89; H, 11.81.
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12-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-ylamino)dodecanoic acid (4)
eluted with hexane/ethyl acetate (1 : 2) to give the crude product,
which was dissolved in chloroform (10 mL). Pyridine (0.12 mL,
1.52 mmol) and bromoacetylbromide (0.13 mL, 1.52 mmol) were
added at 0 ◦C and the mixture was stirred for 4 h at room
temperature. The precipitates were filtered using celite pad and
the filtrate was concentrated in vacuo. The residue was purified
by silica gel column chromatography with hexane/ethyl acetate
(5 : 1) to give 6 as a dark orange solid (0.74 g, 0.88 mmol, 68%):1H
NMR (400 MHz; CDCl3) d 8.51 (d, J = 8.4 Hz, 1H), 6.18 (d,
J = 8.4 Hz, 1H), 5.30 (sext, J = 4.8 Hz, 1H), 4.45–4.24 (m, 2H),
4.20–4.16 (m, 2H), 3.85 (s, 2H), 3.49 (q, J = 12.8, 6.4 Hz, 2H),
2.34–2.31 (m, 4H), 1.81 (quint, J = 7.2 Hz, 2H), 1.61–1.45 (m, 4H),
1.29–1.25 (m, 42H), 0.88 (t, J = 6.4 Hz, 3H); 13C NMR (100 MHz,
CDCl3) d 172.9, 166.5, 166.2, 143.9, 143.6, 136.5, 122.5, 98.2, 68.2,
63.5, 61.4, 43.8, 33.6, 33.0, 31.5, 30.5, 29.3, 29.0, 28.0, 26.6, 25.0,
24.5, 22.3, 13.7; IR (neat) 3333, 3078, 2920, 2855, 1747, 1620,
1350, 1273, 1165, 1119, 1045 cm-1; MS (ESI, positive) m/z 863
([M+Na]+), 879 ([M+K]+); HRMS (ESI, positive) m/z calcd. for
C41H67BrN4O9 [M+Na]+: 861.3989, found: 861.3989.
To a solution of NaHCO3 (4.21 g, 50 mmol) in water (125 mL) was
added an ethanol solution (325 mL) of 12-aminododecanoic acid
(2.70 g, 12.5 mmol) at 37 ◦C and the mixture was stirred for 10 min.
Chloro-7-nitrobenzofurazan (5.02 g, 25 mmol) was added and
then the reaction mixture was stirred at 37 ◦C for overnight with
light shielding. The reaction was quenched by adding HCl (1 M,
56 mL) and the reaction mixture was extracted with chloroform.
The organic layer was washed with water, dried over anhydrous
sodium sulfate, and then concentrated. The residue was purified
by silica gel column chromatography with hexane/ethyl acetate
(20 : 1) to give 4 as an orange solid (2.65 g, 7.01 mmol, 56%):1H
NMR (400 MHz; CDCl3) d 8.51 (d, J = 8.8 Hz, 1H), 6.18 (d, J =
8.4 Hz, 1H), 3.48 (dd, J = 9.2, 15.2 Hz, 2H), 2.36 (t, J = 7.6 Hz, 2H),
1.83–1.47 (m, 4H), 1.30 (s, 14H); 13C NMR (75 MHz, CDCl3) d
178.5, 144.3, 143.9, 136.5, 124.2, 124.0, 98.5, 44.0, 33.7, 29.3, 29.1,
28.9, 28.5, 26.9, 24.6; IR (KBr) 3356, 3244, 2924, 2851, 1709, 1620,
1269, 1188, 1126 cm-1; MS (ESI, positive) m/z 379 ([M+H]+), 401
([M+Na]+), 417 ([M+K]+), 377 ([M - H]-); HRMS (ESI, positive)
m/z calcd. for C18H26N4O5 [M+Na]+: 401.1801, found: 401.1804.
Compound 8
A mixture of 6 (0.94 g, 1.1 mmol) and 7 (0.41 g, 1.1 mmol)34 was
dissolved in anhydrous acetonitrile (12 mL) at room temperature
under argon atmosphere and the mixture was stirred under reflux
for overnight. The solvent was removed under the reduced pressure
and the residue was purified by silica gel column chromatography
with hexane/ethyl acetate (1 : 2) to give 8 as dark orange oil (0.25 g,
(S)-3-(tert-Butyldimethylsilyloxy)-2-(12-(7-nitrobenzo[c][1,2,5]-
oxadiazol-4-ylamino)dodecanoyloxy)propyl stearate (5)
To a mixture of 4 (0.19 g, 0.5 mmol) and N-methylimidazole
(0.12 mL, 1.5 mmol) in dichloromethane (2.6 mL) was added
a dichloromethane solution (1 mL) of tosylchloride (0.14 g,
0.7 mmol) at room temperature under argon atmosphere and the
mixture was stirred for 30 min. A solution of 3 (0.24 g, 0.5 mmol)
in dichloromethane (4 mL) was added to the reaction mixture and
this mixture was stirred at room temperature for overnight. The
reaction was quenched by water and the mixture was extracted
with dichloromethane. The organic layer was washed with brine,
dried over anhydrous Na2SO4, and concentrated. The residue was
purified by silica gel column chromatography with hexane/ethyl
acetate (8 : 1) to give 5 as a dark orange solid (0.23 g, 0.27 mmol,
55%):1H NMR (400 MHz; CDCl3) d 8.51 (d, J = 8.4 Hz, 1H),
6.18 (d, J = 8.8 Hz, 1H), 5.08–5.06 (m, 1H), 4.32 (dd, J = 12.0,
4.0 Hz, 1H), 4.16 (dd, J = 11.6, 6.0 Hz, 1H), 3.72 (d, J = 5.6 Hz,
2H), 3.49 (q, J = 12.4, 7.2 Hz, 2H), 2.31 (ddd, J = 7.2, 2.4 Hz,
4H), 1.81 (quint, J = 6.8 H, 1H), 1.61–1.42 (m, 5H), 1.29–1.25 (m,
42H), 0.88 (s, 12H), 0.05 (s, 6H); 13C NMR (100 MHz, CDCl3)
d 173.4, 173.0, 144.2, 143.8, 136.4, 123.7, 98.4, 71.6, 62.3, 61.3,
43.9, 34.2, 34.1, 31.8, 29.6, 29.3, 29.1, 28.4, 26.8, 25.7, 24.8, 22.6,
18.1, 14.0, -5.5; IR (neat) 3329, 2924, 2855, 1744, 1620, 1350,
1312, 1258, 1157, 1119, 1038 cm-1; MS (ESI, positive) m/z 856
([M+Na]+), 872 ([M+K]+); HRMS (ESI, positive) m/z calcd. for
C45H80N4O8Si [M+Na]+: 855.5643, found: 855.5641.
1
0.25 mmol, 23%): H NMR (400 MHz; CDCl3) d 8.45 (d, J =
8.0 Hz, 1H), 6.28 (d, J = 8.8 Hz, 1H), 5.26–5.25 (m, 1H), 4.37–
3.87 (m, 4H), 3.48 (s, 2H), 3.37–3.27(m, 4H), 3.00–2.97 (m, 2H),
2.32–2.26 (m, 4H), 1.72 (quint, J = 7.2 Hz, 4H), 1.56–1.54 (m, 2H),
1.28–1.24 (m, 42H), 0.86 (t, J = 8.0 Hz, 3H); 13C NMR (100 MHz,
CD3OD) d 174.8, 166.6, 159.2, 146.2, 145.4, 138.7, 122.3, 118.2,
100.0, 69.9, 63.0, 62.7, 44.9, 44.6, 38.5, 34.9, 34.7, 32.8, 30.6,
30.2, 30.0, 27.8, 25.7, 23.5, 16.2, 14.4; IR (neat) 3564, 3329, 3082,
2909, 2851, 1732, 1620, 1180, 1119, 1045, 1003 cm-1; MS (ESI,
negative) m/z 987 ([M - H]-); HRMS (ESI, negative) m/z calcd.
for C45H83B12N5O9S [M - H]-: 988.6950, found: 988.6953.
Fluorescence-labeled closo-dodecaborane lipid (FL-SBL)
Compound 8 (0.15 g, 0.15 mmol) was dissolved in dried THF
(10 mL) and a THF (4 mL) solution of t-BuOK (0.026 g,
0.22 mmol) was added dropwise. The reaction progress was
monitored by thin layer chromatography (TLC). The solvent was
removed under reduced pressure and the residue was purified by
silica gel column chromatography with hexane/ethyl acetate (1 : 2)
to give FL-SBL as dark orange oil (0.075 g, 0.074 mmol, 49%):
1H NMR (400 MHz; CDCl3) d 8.44 (d, J = 8.4 Hz, 1H), 6.29 (d,
J = 8.4 Hz, 1H), 5.20–5.15 (m, 1H), 4.29–4.13 (m, 4H), 3.47–3.41
(m, 2H), 3.21 (s, 2H), 2.43–2.24 (m, 4H), 1.72–1.69 (m, 4H), 1.52
(s, 2H), 1.38–1.21 (m, 42H), 0.84 (t, J = 8.0 Hz, 3H); 13C NMR
(100 MHz, CD3CN) d 174.1, 160.8, 145.5, 145.3, 138.3, 123.7,
122.0, 99.8, 69.8, 63.6, 62.6, 44.6, 36.0, 35.8, 34.7, 34.5, 32.5, 30.3,
29.9, 29.6, 27.5, 25.5, 23.3, 14.3; IR (neat) 3568, 3422, 3321, 3082,
2924, 2855, 1732, 1620, 1269, 1188, 1161, 1123, 1053 cm-1; MS
(ESI, negative) m/z 466 ([(M - 2K])/2]2-); HRMS (ESI, negative)
m/z calcd. for C41H78B12K2N4O9S [(M - 2K)/2]2-: 467.3303, found:
467.3305.
(S)-3-(2-Bromoacetoxy)-2-(12-(7-nitrobenzo[c][1,2,5]oxadiazol-4-
ylamino)dodecanoyloxy)propyl stearate (6)
To a solution of 5 (1.08 g, 1.3 mmol) in THF (10 mL) was added
tetrabutylammonium fluoride (TBAF, 1 M in 1% water containing
THF, 1.4 mL) at room temperature and the mixture was stirred
for 7 h. The mixture was concentrated under the reduced pressure
and dissolved in ethyl acetate. The mixture was washed with water,
dried over anhydrous MgSO4, and then concentrated. The residue
was passed through a short silica gel column chromatography
1378 | Org. Biomol. Chem., 2012, 10, 1374–1380
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Preparation and characterization of the FL-SBL-labeled
PEGylated DSPC liposomes
Rhodamine-conjugated secondary antibody (Santa Cruz). The
samples were coverslipped using Vectashield mounting medium
(Vector Lavoratories, USA) for further analysis under a fluores-
cence confocal microscope (FV100D IX81, OLYMPUS, Japan).
All experiments using mice were performed in compliance with
the relevant laws and institutional guidelines approved by the
Institutional Animal Care and Use Committee in Gakushuin
University.
PEGylated DSPC-liposomes were prepared from DSPC, CH,
and DSPE-PEG-OMe (1 : 1 : 0.11, molar ratio) by the reverse-
phase evaporation (REV) method 30. Briefly, a mixture of DSPC
(158 mg), CH (77 mg), and DSPE-PEG-OMe (64 mg) were
dissolved in 10 ml of chloroform/diisopropylether mixture (1 : 1,
v/v) in a round-bottom flask. To the lipid solution was added
5 mL of water, forming an emulsion. The volume ratio of the
aqueous phase to the organic phase was maintained at 1 : 2.
The emulsion was sonicated for 1 min (SMT Company, UH-
50 Tip-type 50 W, Japan), and then the organic solvent was
removed under vacuum in a rotary evaporator at 37 ◦C with
repeated breaking down of the generated aggregate to obtain a
suspension of liposomes. The liposomes obtained were subjected
to extrusion 10 times through a polycarbonate membrane of
100 nm pore size (Whatman, 110605, FILTER, 0.1UM, 25
MM, Gentaur Molecular Products, Belgium), using an extruder
device (Lipex Biomembrane, Canada) thermostatted at 60 ◦C.
Purification was accomplished by ultracentrifuging at 200,000
g for 20 min at 4 ◦C (himac cp 80 wx, Hitachi Koki, Japan)
to obtain PEGylated DSPC liposomes as a pellet. FL-SBL was
dissolved in normal saline solution and various volumes (10–
40 mL) of the FL-SBL solution (10 mg mL-1) were mixed with
500 mL of DSPC liposome solution described above (ca. 2000
ppm P concentration). The mixture was maintained at 25 ◦C
for 5 min and free FL-SBL was removed by ultracentrifugation
at 200,000 g for 60 min at 4 ◦C. The obtained FL-SBL-labeled
liposomes were resuspended in PBS or water. Liposome size
and zeta potential were measured with an electrophoretic light
scattering spectrophotometer (Nano-ZS, Sysmex, Japan). The
compositions of FL-SBL and DSPC in liposomes were calculated
from data obtained by the simultaneous measurement of boron
and phosphorus concentrations by inductively coupled plasma
atomic emission spectroscopy (ICP-AES, HORIBA, Japan). The
fluorescence intensity of each sample was measured at a range
of emission wavelengths from 475 to 650 nm using an excitation
wavelength of 460 nm.
Acknowledgements
We thank Professor Yoshiyuki Inaguma (Gakushuin University)
for the measurement of concentration-dependent increase of
fluorescent intensity of the FL-SBL-labeled PEGylated DSPC
liposomes. This work was supported in part by Health and Labour
Sciences Research Grants for Research on Nanotechnical Medical
from the Ministry of Health, Labour and Welfare (20100201) and
the Ministry of Education, Science, Sports, Culture and Technol-
ogy, Grant-in-Aid for Scientific Research (B) (No. 23390018) from
Japan.
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