Liposomal Encapsulation of Other Boranes and Carboranes

Polyhedral boranes34 and carboranes113,114 are another class of boron compounds that have been used for NCT. They contain multiple boron atoms per molecule, are resistant to metabolic degradation, and are lipophilic, thereby permitting easier penetration of the tumor cell membrane.113 In addition to BSH, carboranylpropylamine (CPA, Figure 6.7, 4)115 has been incorporated into conventional and PEGylated liposomes by active loading, using a transmembrane pH gradient.115,116 Although as many as 13,000 molecules of CPA were loaded into liposomes having a mean average diameter of 100 nm, there was no in vitro toxicity to both the glioblastoma cell line SK-MG-1 and normal human peripheral blood lymphocytes. Borane anions, such as B10H2K, B12H11SH2k, B20H17OH4k, and B20H3K, and the normal form and photoisomer of B20h1k, also have been encapsulated into small unilamellar vesicles with mean diameters of 70 nm or less.117-120 These were composed of the pure synthetic phospholipids, distearoyl phosphatidylcholine, and cholesterol.117 Although encapsulation efficiencies were only 2-3%, following i.v. injection, these liposomes were selectively delivered to the tumors in mice bearing the EMT6 mammary carcinoma and had attained boron concentrations of greater than 15 mg boron/g, with a T:Bl ratio of greater than 3. Two isomers of B20h1k attained boron concentrations of 13.6 and 13.9 mg/g with T:Bl ratios of 3.3 and 12, respectively, at 48 h following administration. High boron retention and prolongation of their circulation time was observed due to the interaction with intracellular components after it had been released from liposomes within tumor cells.117

To examine the effect of charge and substitution on the retention of boranes, two isomers [B20H17NH3]3K and [1-(2'-B10H9)-2-NH3B10H8]3k (5) were prepared from the polyhedral




ClH3N(CH2)4 HN(CH2)3

ClH3N(CH2)4 HN(CH2)3

FIGURE 6.7 Structures of hydrophilic and lipophilic boron-containing compounds that have been incorporated into liposomes. Carboranylpropylamine (CPA, 4), [1-(2'-B10H9)-2-NH3B10H8]3k (5), [n-B20H18]2~ (6), [B20H17SH]4k (7), Na3[a2-B20H17NH2CH2CH2NH2] (9) and boronated water soluble acridine (WSA, 11) were encapsulated into the aqueous core. K[nido-7-CH3(CH2)15-7,8-C2B9H11] (8) and cholesteryl 1,12-dicarba-closo-dodecaboranel-carboxylate (10) were incorporated into the lipid bilayer of liposomes.

borane anion [n-B20H18]2K (6).118'119 The sodium salts of these two isomers had been encapsulated within small unilamellar liposomes, composed of distearoyl phosphatidylcholine/cholesterol at a 1:1 ratio. Both isomers of [B20H17NH3]3k had excellent tumor uptake and selectivity in EMT 6 tumor bearing mice, even at very low I.D.s, and this resulted in peak tumor boron concentrations of 30-40 mg B/g and a T:Bl ratio of approximately 5. Due to low boron retention of liposomal Na3[B20H19] and Na4[e2-B20H17OH] and rapid clearance of liposomal [2-NH3B10H9]K, the enhanced retention of liposomal Na3[ae-B20H17NH3] was not due to the anionic charge or substitution in the borane cage. Rather, it could be attributed to their facile intracellular oxidation to an extremely reactive NH3-substituted [n-B20H18]2K electrophilic anion, [B20H17NH3]K. Another anion [ae-B20H17NH3]3 K also was encapsulated into liposomes prepared with 5% PEG-2000-distearoyl phosphatidylethanolamine as a constituent of the membrane. These liposomes had longer in vivo circulation times, which resulted in continued accumulation of boron in the tumor over the entire 48 h time period, and reached a maximum concentration of 47 mg B/g tumor.

[B20H17SH]4k (7), a thiol derivative of [B20H18]4k, possesses a reactive thiol substituent and this can be oxidized into the more reactive [B20H17SH]2k anion. Both of these were considered to be essential for high tumor boron retention120 and they have been encapsulated into small, unilamellar liposomes. Biodistribution was determined after i.v. injection into BALB/c mice bearing EMT6 tumors. At low I.D.s, tumor boron concentrations increased throughout the duration of the experiment, resulting in a maximum concentration of 47 mg B/g tumor at 48 h, which corresponded to 22.2% I.D./g and a T:Bl ratio of 7.7. This was the most promising of the polyhedral borane anions that had been investigated for liposomal delivery. Although they were able to deliver adequate amounts of boron to tumor cells, their application to BNCT has been limited due to their low incorporation efficiency (approximately 3%).

Lipophilic boron compounds incorporated into the lipid bilayer would be an alternative approach. Small unilamellar vesicles composed of 3:3:1 ratio of distearoylphosphatidylcholine, cholesterol and K[nido-7-CH3(CH2)15-7,8-C2B9H11] (8) in the lipid bilayer and Na3[a2-B20H17 NH2CH2CH2NH2] (9) in the aqueous core were produced as a delivery agents for NCT mediated synovectomy.121 Biodistribution studies were carried out in Louvain rats that had a collagen-induced arthritis. The maximum synovial boron concentration was 29 mg/g tissue at 30 h and this had only decreased to 22 mg/g at 96 h following i.v. administration. The prolonged retention by synovium provided sufficient time for extensive clearance of boron from other tissues so that at 96 h the synovium to blood (Syn:Bl) ratio was 3.0. To accelerate blood clearance, serum stability of the liposomes was lowered by increasing the proportion of K[nido-7-CH3(CH2)15-7,8-C2B9H11] embedded in the lipid bilayer. Liposomes were formulated with a 3:3:2 ratio of DSPC to Ch to K[nido-7-CH3(CH2)15-7,8-C2B9H11] in the lipid bilayer and Na3[a2-B20H17NH2CH2CH2NH2] was encapsulated in the aqueous core. The boron concentration in the synovium reached a maximum of 26 mg/g at 48 h with a Syn:Bl ratio of 2, following which it slowly decreased to 14 mg/g at 96 h at which time the Syn:Bl ratio was 7.5.121

Another method to deliver hydrophilic boron containing compounds would be to incorporate them into cholesterol to target tumor cells expressing amplified low density lipoprotein (LDL)


receptors. Glioma cells, which absorb more cholesterol, have been reported to take up more

LDL than the corresponding normal tissue cells.125_127 The cellular uptake of liposomal cholesteryl 1,12-dicarba-closo-dodecaboranel-carboxylate (10) by two fast growing human glioma cell lines, SF-763 and SF-767, was mediated via the LDL receptor and was much higher than that of human neurons. The cellular boron concentration was approximately 10_11 times greater than that required for BNCT.128

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