Spermidinium closo-dodecaborate-encapsulating liposomes as efficient boron delivery vehicles for neutron capture therapy

Article abstract of DOI:10.1039/c4cc04344h

closo-Dodecaborate-encapsulating liposomes were developed as boron delivery vehicles for neutron capture therapy. The use of spermidinium as a counter cation of closo-dodecaborates was essential not only for the preparation of high boron content liposome solutions but also for efficient boron delivery to tumors. This journal is

Full text of DOI:10.1039/c4cc04344h

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Spermidinium closo-dodecaborate-encapsulating
liposomes as efficient boron delivery vehicles for
neutron capture therapy†
Cite this: Chem. Commun., 2014,
0, 12325
5
Received 9th June 2014,
Accepted 21st August 2014
ab
b
b
bc
Shoji Tachikawa, Tatsuro Miyoshi, Hayato Koganei, Mohamed E. El-Zaria,
d
e
e
ab
Clara Vinas, Minoru Suzuki, Koji Ono and Hiroyuki Nakamura*
˜
DOI: 10.1039/c4cc04344h
www.rsc.org/chemcomm
10–12
closo-Dodecaborate-encapsulating liposomes were developed as boron bilayer have been studied.
Hawthorne and coworkers developed
delivery vehicles for neutron capture therapy. The use of spermidinium liposomes incorporating Na [1-(2-B H )-2-NH B H ] into the internal
3
10
9
3 10 8
as a counter cation of closo-dodecaborates was essential not only for aqueous core and K[nido-7-CH (CH ) -7,8-C B H ] into the bilayer
3
2 15
2 9 11
8b,13
the preparation of high boron content liposome solutions but also for membrane to increase the boron content in liposomes.
efficient boron delivery to tumors.
2
We previously developed Na BSH-encapsulating 10% distearoyl
11b
boron lipid (DSBL) liposomes that have high boron content with
Boron neutron capture therapy (BNCT) has been attracting growing
interest as one of the minimally invasive cancer therapies.
Mercaptoundecahydrododecaborate (Na [B12H11SH]; Na BSH)
2 2
and L-p-boronophenylalanine (L-BPA) have been used in BNCT
14
excellent boron delivery efficacy to tumors. In this communication,
1
we studied the effects of the counter cations of boron clusters on
liposome formation to develop high boron content liposomes for
BNCT by overcoming osmotic pressure limitations.
for many years. L-BPA, in particular, has been widely used for the
We selected three closo-dodecaborates, Na [B H ],
2
12 12
2
treatment of not only melanoma but also brain tumor and head
15
16
Na
Fig. 1). We first tested the cytotoxicity of the closo-dodecaborates
toward colon 26 cells. The closo-dodecaborates are relatively non-
toxic and the GI50 values of Na 12], Na BSH, Na[B12 ],
and Na [B12 11OH] are 5.1, 2.1, 32.9, and 7.7 mM, respectively
Table S1 in the ESI†). Liposomes containing the closo-
2
[B12
H
11OH] and Na[B12
H
11NH
3
]
2
in addition to Na BSH
3
and neck cancer because it can be taken up selectively by tumor
(
4
cells through an amino acid transporter. The accelerator-based
BNCT is now undergoing phase I clinical study for the treatment
2
[B12
H
2
H11NH
3
5
,6
of brain tumor and head and neck cancer patients in Japan.
2
H
1
0
In recent years, liposomal B carriers have attracted attention
(
7
–10
as some of the efficient boron delivery systems in BNCT.
Several efficient in vivo BNCTs have been reported. Yanagi ¨e and
coworkers demonstrated the first antitumor effect of Na BSH-
encapsulating liposomes conjugated with a monoclonal antibody
dodecaborates were prepared from DSPC, cholesterol, and
DSPE-PEG2000 by the reverse phase evaporation method with
sizes of approximately 100 nm in diameter. The results are
summarized in Table 1. The final boron and phosphorus con-
2
9
a,b
specific for the carcinoembryonic antigen.
co-workers developed transferrin-conjugating Na
Maruyama and
BSH-encapsulating
2
centrations of liposome solution containing Na [B12H12] were
2
3
438 Æ 2.0 and 2864 Æ 18.3 ppm, respectively, and the B/P ratio
9d,10
liposomes.
Although they succeeded in completely suppressing
was 1.2 (entry 1). The higher B/P ratio indicates the higher boron
content in liposomes. The liposome yield was 58% based on the
total phospholipids used in the preparation. The B/P ratio of the
liposome containing Na[B H NH ] was 2.2 (entry 2), which was
tumor growth in mice after neutron irradiation, the concentration of
10
inner B of liposomes was limited in preparation due to osmotic
reasons. For this reason, boron lipids embedded within the liposome
1
2
11
3
slightly higher than those of liposomes containing Na
Na 11OH], and Na 12] (1.2–1.6, entries 1, 3, and 4).
In the case of Na[B12 11NH ], an ammonium ion group served
2
BSH,
a
Chemical Resources Laboratory, Tokyo Institute of Technology,
2
[B12H
2
[B12H
4
259 Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan.
H
3
E-mail: hiro@res.titech.ac.jp; Fax: +81 45 924 5244
b
as one of the two counter cations of the closo-dodecaborate
Department of Chemistry, Faculty of Science, Gakushuin University, Mejiro,
Toshima-ku, Tokyo 171-8588, Japan
c
Department of Chemistry, Faculty of Science, Tanta University, 31527-Tanta, Egypt
Institut de Ci `e ncia de Materials de Barcelona (ICMAB-CSIC), Campus U.A.B.,
d
0
8193 Bellaterra, Barcelona, Spain
e
Particle Radiation Oncology Research Center, Kyoto University Research Reactor
Institute, Asashiro-nishi, Kumatori-cho, Sennan-gun, Osaka 590-0494, Japan
Electronic supplementary information (ESI) available: Experimental details and
†
data. See DOI: 10.1039/c4cc04344h
Fig. 1 Structures of closo-dodecaborates used for encapsulation in liposomes.
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Table 1 Physical characteristics of liposomes containing closo-dodecaborates
a
associated with sodium and various ammonium cations
b,c
d
B conc.
(ppm)
P conc.
(ppm)
Yield
(%)
b,c
Entry Boron cluster
B/P
1
2
3
4
5
6
7
8
9
Na
Na[B12
2
BSH
3438 Æ 2.0
2864 Æ 18.3 1.2
58
44
39
47
54
44
98
87
95
H
11NH
11OH]
3
]
4072 Æ 22.8 1835 Æ 38.5 2.2
2635 Æ 184.2 1600 Æ 99.0 1.5
3133 Æ 10.3 1932 Æ 13.3 1.6
2874 Æ 47.7 2225 Æ 15.8 1.3
Na
Na
2
[B12
[B12
H
H
NH
2
12
]
3
(n-C
(H NC
spd-BSH
3
H
7
)
2
BSH
3
4
H
8
NH
3
)BSH 4711 Æ 17.4 1833 Æ 43.4 2.6
13 867 Æ 185.8 4046 Æ 18.3 3.4
(Sperminium)BSH
spd-[B12 11NH
9759 Æ 139.6 3559 Æ 44.5 2.7
13 970 Æ 216.5 3943 Æ 43.4 3.5
Fig. 2 Effect of the amount of spd cation on spd-BSH encapsulation in
H
3
]
liposomes. Boron/phosphorus (B/P) ratios of (spd) -closo-dodecaborate-
x
encapsulating liposomes are shown in the vertical axis.
reached a maximum of 3.4 when the BSH : spd ratio was 1 : 1.
Liposome yields showed a similar tendency to B/P ratios. The highest
liposome yield was observed at the BSH to spd cation ratio of 1 : 1.
a
In all cases, liposomes were prepared from DSPC, cholesterol, and DSPE-
b
PEG2000 (1 : 1 : 0.11, molar ratio) by the REV method. Data are expressed
as means Æ standard deviation (SD). Boron and phosphorus concentra-
c
d
tions of liposome solution were determined by ICP-AES. Liposome yields Transmission electron microscopy (TEM) analysis of spd-BSH-
were calculated from the phosphorus concentration of liposome solution
based on the total phospholipids used in preparation.
2
encapsulating liposomes and Na BSH-encapsulating liposomes
was also carried out using Cryo-TEM (Fig. 3). It is notable that
the liposomes interacted with each other in the case of Na BSH-
2
encapsulating liposomes, whereas the liposomes dispersed in
solution without interacting with each other in the case of spd-
BSH-encapsulating liposomes.
dianion. We speculated that ammonium counter cations would
affect the encapsulation of closo-dodecaborates in liposomes.
2
Recently, Gabel and coworkers reported that Na BSH induces
We next examined boron distribution of the spd-closo-
dodecaborate-encapsulating liposomes in colon 26 tumor-bearing
aggregation and membrane rupture, increasing wall thickness of
the liposome and triggering the release of liposome contents.
Indeed, it is known that tetramethylammonium (TMA) salts of
closo-dodecaborates are insoluble in water, and the ion-exchange
1
7
18
mice. The liposomes were injected at doses of 15, 30, and 100 mg
À1
[
B] kg body weight via the tail veins. Na
2
BSH- and Na
2
[B12
3
H11NH ]-
encapsulating liposomes were also injected at a dose of
from Na
exchange from (TMA)
2
BSH to (TMA)
BSH to Na
2
BSH proceeds readily, whereas the ion-
À1
3
0 mg [B] kg as control experiments. The time courses of boron
2
2
BSH is not easy. We predicted
distribution in each organ are shown in Fig. 4. Blood boron
concentrations of 460.7, 104.0, and 33.2 ppm were detected 24 h
that encapsulation as well as liposome yield would be increased
if we could reduce this interaction in the preparation of closo-
dodecaborate-encapsulating liposomes. Thus we prepared various
ammonium salts of BSH and examined their encapsulation into
liposomes (entries 5–9 in Table 1). The B/P ratio of the liposome
after injection of spd-BSH-encapsulating liposomes (100, 30, and
À1
1
5 mg [B] kg ), respectively (Fig. 4a). Blood boron concentration in
À1
mice injected with 100 mg [B] kg of spd-BSH-encapsulating
+
liposomes did not decrease notably during the 48 h period, whereas
containing the n-C H NH salt of BSH was similar to that of the
3
7
3
À1
+
+
those in mice injected with 30 and 15 mg [B] kg of spd-BSH-
liposome containing Na
cation increased the B/P ratio (2.6) and boron concentration
4711 ppm). Interestingly, the B/P ratio dramatically increased to
.4 when the spermidinium (spd) cation was employed (entry 7).
2 3 4 8 3
BSH (entries 1 vs. 5). The H N C H NH
encapsulating liposomes gradually decreased in a time-dependent
manner. The time courses of boron concentrations in liver, kidneys,
and spleen are shown in Fig. 4b–d, respectively. Boron concentra-
(
3
tions of 528.5, 144.2, and 74.4 ppm in liver were observed 48 h after
In addition, the liposome yield was markedly increased to 98% and
the final boron concentration of the liposome solution reached
À1
the injection of 100, 30, and 15 mg [B] kg
of spd-BSH-
encapsulating liposomes, respectively. In the meantime, maximum
13 867 ppm. In contrast, the B/P ratio of liposome containing
(sperminium) BSH dropped to 2.7, although the liposome yield
was still high (87% yield) and the final boron concentration of the
liposome solution was high at 9759 ppm (entry 8). Liposome
containing spd-[B12H11NH ] showed the highest B/P ratio (3.5); the
3
boron concentration of the liposome solution reached 13 790 ppm
and the liposome yield was 95% (entry 9).
We examined whether the formation of high boron content
liposomes is affected by the viscosity of the closo-dodecaborate
2À
solutions. However, the spd cation of [B H ] does not affect the
1
2 12
viscosity of internal aqueous solution of liposomes (Table S3, ESI†).
We measured liposome yields and B/P ratios under the condition of
2
various ratios of BSH to spd cations (Na BSH: [spermidine + HCl] = 1:
2
Fig. 3 TEM images of Na BSH-encapsulating liposomes (left) and spd-
X, X = 0, 0.25, 0.5, 1, and 2). As shown in Fig. 2, the B/P ratio BSH-encapsulating liposomes (right). Scale bar represents 200 nm.
1
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Fig. 5 Tumor volumes in mice (Balb/c, female, six weeks old, 14–20 g)
bearing colon 26 solid tumor, exposed to thermal neutron irradiation (hot)
Fig. 4 Time courses of distribution of spd-BSH- and Na
2
BSH-encapsulating
12
2
for 50 min (1.3–2.2 Â 10 neutrons per cm ) or not exposed to thermal
À1
À1
liposomes (n, spd-BSH: 15 mg [B] kg ; B, spd-BSH: 30 mg [B] kg ; J,
neutron irradiation (cold). Irradiation was performed 36 h after injection of
À1
À1
spd-BSH: 100 mg [B] kg ; m, Na BSH: 15 mg [B] kg ). Each liposome
2
1
0
10
À1
10
liposomes containing spd-[ BSH] (n, 15;B, 30; J, 100 mg [ B] kg ) and
was injected into tumor-bearing mice via the tail vein. Data are expressed as
means Æ SD (n = 5).
10
10
2 2
À1
Na [ BSH] (E , 30 mg [ B] kg ), or 1 h after injection of Na [ BSH]
10
À1
solution (’, 100 mg [ B] kg ). K, hot control; Â, cold control. **P o 0.01,
compared with hot control.
tumor boron concentrations of 202.7 and 82.4 ppm were achieved
À1
10
À1
3
6 h after injection at doses of 100 and 30 mg [B] kg , respectively. also inhibited tumor growth at a dose of 30 mg [ B] kg , and the
À1
Even at the low boron dose of 15 mg [B] kg , the tumor boron tumor was completely controlled three weeks after thermal neutron
concentration was 34.0 ppm at 36 h after injection (Fig. 4e). We irradiation. Tumor growth was suppressed in mice treated with
10
10
À1
also demonstrated the boron distribution of Na
2
BSH-encapsulating Na [ BSH] solution (100 mg [ B] kg ) during the two weeks after
2
liposomes in tumor-bearing mice for comparison. Although thermal neutron irradiation. However, the tumor started to grow
blood and liver boron concentrations after injection of Na BSH- thereafter (Fig. 5). In contrast, tumor growth was not suppressed in
2
À1
10
10
À1
3
encapsulating liposomes at a dose of 30 mg [B] kg were similar mice treated with Na[ B12H11NH ] solution (100 mg [ B] kg )
to those after injection of spd-BSH-encapsulating liposomes at a even after thermal neutron irradiation (Fig. S2, ESI†).
À1
dose of 30 mg [B] kg , kidney and spleen boron concentrations
Fig. 6 shows the survival curve of tumor-bearing mice after
after injection of Na
2
BSH-encapsulating liposomes were lower thermal neutron irradiation. All untreated mice without thermal
than those after injection of spd-BSH-encapsulating liposomes neutron irradiation died within two weeks. Thermal neutron irradia-
up to 48 h. The tumor boron concentration at 36 h after injecting tion enhanced mouse survival and all mice exposed to thermal
Na
clearance of Na BSH-encapsulating liposomes was slow (Fig. 4e). observed in mice injected with spd-[ BSH]- and spd-[ B12
2
BSH-encapsulating liposomes was 31.9 ppm, although the neutron irradiation died within 78 days. Prolonged survival was
10
10
H11NH ]-
3
2
A similar tendency was observed in spd-[B H NH ]-encapsulating encapsulating liposomes; 72% of the mice that received a dose of
1
2
11
3
1
0
À1
liposomes (Fig. S1, ESI†). Blood, kidney, and spleen boron concentra- 15 mg [ B] kg survived up to 100 days after the thermal
tions gradually decreased after injection. Maximum tumor boron neutron irradiation. Furthermore, a remarkable antitumor effect
concentrations of 242.2, 88.7, and 35.4 ppm were achieved 6 h after
À1
injection at doses of 100, 30, and 15 mg [B] kg , respectively.
Interestingly, significant tumor boron accumulation was also observed
in the case of Na[B12H11NH ]-encapsulating liposomes.
3
Finally, we examined the antitumor effect of liposomes
containing spd closo-dodecaborates in colon 26 tumor bearing
mice exposed to thermal neutron irradiation. Thermal neutron
irradiation of the tumor-transplanted left thighs of mice was
carried out 36 h after injection. The tumor growth curves of
mice are shown in Fig. 5 (and in Fig. S2, ESI†). ‘‘Hot control
(
–K–)’’ and ‘‘Cold control (––)’’ represent tumor volumes of mice
injected with saline with and without thermal neutron irradiation,
respectively. Tumor growth was significantly suppressed in mice
treated with spd-[ BSH]- and spd-[ B H NH ]-encapsulating
liposomes at doses of 15, 30, and 100 mg [ B] kg and exposed
to thermal neutron irradiation. The tumor completely disappeared
within three weeks even when a dose of 15 mg [ B] kg was
10
10
Fig. 6 Survival curve of tumor-bearing mice after thermal neutron irradiation.
The irradiation was performed 36 h after injection of closo-dodecaborates
12
11
10
3
À1
1
0
À1
À1
10
À1
(
TT, spd-BSH: 30 mg
[
B] kg ; E, spd-BSH: 15 mg
[
B] kg ; B,
10
10
12
spd-
B H
12
11NH
3
: 15 mg [ B] kg ) for 50 min (1.3–2.2 Â 10 neutrons per
10
À1
2
cm ). Â, cold control; K, hot control. Mice were sacrificed when their tumor
10
10
3
employed. Liposomes containing Na
2
[ BSH] and Na[ B12
3
H11NH ]
volumes reached B3000 mm .
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was observed in the mice treated with spd-[ BSH]-encapsulating
liposomes at a dose of 30 mg [ B] kg ; 100% of the mice
survived up to 100 days after the thermal neutron irradiation.
We succeeded in the preparation of high boron content lipo-
somes. The use of spd as a counter cation of closo-dodecaborates was
essential to obtain the liposomes with high yields and high B/P
ChemComm
1
0
6 H. Tanaka, Y. Sakurai, M. Suzuki, T. Takata, S. Masunaga,
Y. Kinashi, G. Kashino, Y. Liu, T. Mitsumoto, S. Yajima,
H. Tsutsui, M. Takada, A. Maruhashi and K. Ono, Appl. Radiat. Isot.,
1
0
À1
2
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8
9
For passive targeting, see: (a) K. Shelly, D. A. Feakes,
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1
0
À1
10
ratios. All of the mice injected with 30 mg [ B] kg of spd-[ BSH]-
encapsulating liposomes were completely cured while five of seven
1
0
À1
10
mice injected with 15 mg [ B] kg
of spd-[ BSH]- and
1
0
spd-[ B12H11NH ]-encapsulating liposomes were cured 100 days
3
after thermal neutron irradiation. The results indicate that the
total amount of phospholipids could be reduced to less than
1
0
one-seventh of those used to prepare Na [ BSH]-encapsulating
2
1
9
liposomes.
We believe that the spd-closo-dodecaborate-
encapsulating liposomes are promising candidates for clinical
use in BNCT.
We thank Dr S. Kondoh (Teijin Pharm. Co. Ltd) for the
measurement of osmotic pressures and Professor K. Iwata
1
1
(Gakushuin University) for the measurement of the viscosity.
This work was supported by Health and Labor Sciences Research
Grants for research on Nanotechnical Medical from the Ministry
of Health, Labor and Welfare (20100201) and the Ministry of
Education, Science, Sports, Culture and Technology, Grant-in-
Aid for Scientific Research (B) (No. 23390018) from Japan.
1
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W. Tjarks and R. J. Lee, Bioconjugate Chem., 2007, 18, 101.
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Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 6512.
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1
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5
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