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

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.

Boron neutron capture therapy (BNCT) has been attracting growing interest as one of the minimally invasive cancer therapies. 1Mercaptoundecahydrododecaborate (Na 2 [B 12 H 11 SH]; Na 2 BSH) and L-p-boronophenylalanine (L-BPA) have been used in BNCT for many years.L-BPA, in particular, has been widely used for the treatment of not only melanoma but also brain tumor 2 and head and neck cancer 3 because it can be taken up selectively by tumor cells through an amino acid transporter. 4The accelerator-based BNCT is now undergoing phase I clinical study for the treatment of brain tumor and head and neck cancer patients in Japan. 5,6[9][10] Several efficient in vivo BNCTs have been reported.Yanagie ¨and coworkers demonstrated the first antitumor effect of Na 2 BSHencapsulating liposomes conjugated with a monoclonal antibody specific for the carcinoembryonic antigen.9a,b Maruyama and co-workers developed transferrin-conjugating Na 2 BSH-encapsulating liposomes.9d,10 Although they succeeded in completely suppressing tumor growth in mice after neutron irradiation, the concentration of inner 10 B of liposomes was limited in preparation due to osmotic reasons.1][12] Hawthorne and coworkers developed liposomes incorporating Na 3 [1-(2-B 10 H 9 )-2-NH 3 B 10 H 8 ] into the internal aqueous core and K[nido-7-CH 3 (CH 2 ) 15 -7,8-C 2 B 9 H 11 ] into the bilayer membrane to increase the boron content in liposomes.8b,13 We previously developed Na 2 BSH-encapsulating 10% distearoyl boron lipid (DSBL) 11b liposomes that have high boron content with excellent boron delivery efficacy to tumors. 14In this communication, 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.
We selected three closo-dodecaborates, Na 2 [B 12 H 12 ], Na 2 [B 12 H 11 OH] 15 and Na[B 12 H 11 NH 3 ] 16 in addition to Na 2 BSH (Fig. 1).We first tested the cytotoxicity of the closo-dodecaborates toward colon 26 cells.The closo-dodecaborates are relatively nontoxic and the GI 50 values of Na   S1 in the ESI †).Liposomes containing the closododecaborates 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 concentrations of liposome solution containing Na 2 [B 12 H 12 ] were 3438 AE 2.0 and 2864 AE 18.3 ppm, respectively, and the B/P ratio 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 12 H 11 NH 3 ] was 2.2 (entry 2), which was slightly higher than those of liposomes containing Na 2 BSH, Na 2 [B 12 H 11 OH], and Na 2 [B 12 H 12 ] (1.2-1.6,entries 1, 3, and 4).In the case of Na[B 12 H 11 NH 3 ], an ammonium ion group served as one of the two counter cations of the closo-dodecaborate  dianion.We speculated that ammonium counter cations would affect the encapsulation of closo-dodecaborates in liposomes.Recently, Gabel and coworkers reported that Na 2 BSH induces aggregation and membrane rupture, increasing wall thickness of the liposome and triggering the release of liposome contents. 17Indeed, it is known that tetramethylammonium (TMA) salts of closo-dodecaborates are insoluble in water, and the ion-exchange from Na 2 BSH to (TMA) 2 BSH proceeds readily, whereas the ionexchange from (TMA) 2 BSH to Na 2 BSH is not easy.We predicted that encapsulation as well as liposome yield would be increased if we could reduce this interaction in the preparation of closododecaborate-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 containing the n-C 3 H 7 NH 3 + salt of BSH was similar to that of the liposome containing Na 2 BSH (entries 1 vs. 5).The H 3 N + C 4 H 8 NH 3 + cation increased the B/P ratio (2.6) and boron concentration (4711 ppm).Interestingly, the B/P ratio dramatically increased to 3.4 when the spermidinium (spd) cation was employed (entry 7).
In addition, the liposome yield was markedly increased to 98% and the final boron concentration of the liposome solution reached 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-[B 12 H 11 NH 3 ] showed the highest B/P ratio (3.5); the 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 solutions.However, the spd cation of [B 12 H 12 ] 2À does not affect the viscosity of internal aqueous solution of liposomes (Table S3, ESI †).We measured liposome yields and B/P ratios under the condition of various ratios of BSH to spd cations (Na 2 BSH: [spermidine + HCl] = 1: X, X = 0, 0.25, 0.5, 1, and 2).As shown in Fig. 2, the B/P ratio 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. Transmission electron microscopy (TEM) analysis of spd-BSHencapsulating liposomes and Na 2 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 2 BSHencapsulating liposomes, whereas the liposomes dispersed in solution without interacting with each other in the case of spd-BSH-encapsulating liposomes.
We next examined boron distribution of the spd-closododecaborate-encapsulating liposomes in colon 26 tumor-bearing mice. 18The liposomes were injected at doses of 15, 30, and 100 mg        was observed in the mice treated with spd-[ 10 BSH]-encapsulating liposomes at a dose of 30 mg [ 10 B] kg À1 ; 100% of the mice survived up to 100 days after the thermal neutron irradiation.
We succeeded in the preparation of high boron content liposomes.The use of spd as a counter cation of closo-dodecaborates was essential to obtain the liposomes with high yields and high B/P ratios.All of the mice injected with 30 mg [ 10 B] kg À1 of spd-[ 10 BSH]encapsulating liposomes were completely cured while five of seven mice injected with 15 mg [ 10 B] kg À1 of spd-[ 10 BSH]-and spd-[ 10 B 12 H 11 NH 3 ]-encapsulating liposomes were cured 100 days after thermal neutron irradiation.The results indicate that the total amount of phospholipids could be reduced to less than one-seventh of those used to prepare Na 2 [ 10 BSH]-encapsulating liposomes. 19We believe that the spd-closo-dodecaborateencapsulating liposomes are promising candidates for clinical use in BNCT.

a
Chemical Resources Laboratory, Tokyo Institute of Technology, [B] kg À1 body weight via the tail veins.Na 2 BSH-and Na 2 [B 12 H 11 NH 3 ]encapsulating liposomes were also injected at a dose of 30 mg [B] kg À1 as control experiments.The time courses of boron 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 after injection of spd-BSH-encapsulating liposomes (100, 30, and 15 mg [B] kg À1), respectively (Fig.4a).Blood boron concentration in mice injected with 100 mg [B] kg À1 of spd-BSH-encapsulating liposomes did not decrease notably during the 48 h period, whereas those in mice injected with 30 and 15 mg [B] kg À1 of spd-BSHencapsulating 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 concentrations of 528.5, 144.2, and 74.4 ppm in liver were observed 48 h after the injection of 100, 30, and 15 mg [B] kg À1 of spd-BSHencapsulating liposomes, respectively.In the meantime, maximum

a
In all cases, liposomes were prepared from DSPC, cholesterol, and DSPE-PEG2000 (1 : 1 : 0.11, molar ratio) by the REV method.b Data are expressed as means AE standard deviation (SD).c Boron and phosphorus concentrations of liposome solution were determined by ICP-AES.d Liposome yields were calculated from the phosphorus concentration of liposome solution based on the total phospholipids used in preparation.

Fig. 2
Fig. 2 Effect of the amount of spd cation on spd-BSH encapsulation in liposomes.Boron/phosphorus (B/P) ratios of (spd) x -closo-dodecaborateencapsulating liposomes are shown in the vertical axis.

Table 1
Physical characteristics of liposomes containing closo-dodecaborates associated with sodium and various ammonium cations a