The development of lipophilic boron compounds embedded within the liposome bilayer is an attractive means to increase the overall incorporation efficiency of boron-containing species, as well as to raise the gross boron content of the liposomes in the formulation. In 1991, BSH encapsulated in a liposome which was conjugated with mAb specific for carcinoembryonic antigen (CEA) was investigated as a liposomal delivery system. This immunoliposome was shown to bind selectively to human pancreatic carcinoma cells (AsPC-1) bearing CEA on their surfaces, and to inhibit tumor cell growth on thermal neutron irradiation in vitro. Furthermore, the cytotoxic effects of locally injected BSH-encapsulated immunoliposomes on AsPC-1 xenografts in nude mice were evaluated. After i.t. injection of the immunoliposomes, boron concentrations in tumor tissue and blood were about 50 ± 6 and about 0.3 ± 0.1 ppm, respectively. Tumor growth in mice that received an i.t. injection of BSH-encapsulated immunoliposomes was suppressed with thermal neutron irradiation in vivo. In 1992, boron-encapsulated liposomes with mean diameters of 70 nm or less from distearoylphosphatidylcholine (DSPC) and cholesterol were prepared. Hydrolytically stable borane anions, B₁₀H₁₀²⁻, B₁₂H₁₁SH²⁻, B₂₀H₁₇OH⁴⁻, B₂₀H₁₉³⁻ and B₂₀H₁₈²⁻ (two isomers), were encapsulated in the liposomes as soluble sodium salts. The highest boron concentrations in tumor reached the therapeutic range (>15 μg of boron per g of tumor) while maintaining high tumor/blood ratios (>3). The most favorable results were obtained with the two isomers of B₂₀H₁₈²⁻. These boron compounds have the ability to react with intracellular components after they have been deposited in tumor cells by liposomes, thereby preventing the release of the boron compounds into blood. Selective boron delivery to tumors by lipophilic species incorporated in the membranes of unilamellar liposomes was demonstrated by Hawthorne and coworkers in 1995 . They synthesized a nido-carborate amphiphile (K[nido-7-CH₃(CH₂)₁₅-7,8-C₂B₉H₁₁], MAC) and prepared boronated liposomes composed of distearoylphosphatidylcholine (DSPC), cholesterol, and MAC in the bilayer. After injecting liposomal suspensions into BALB/c mice bearing EMT6 mammary adenocarcinomas, the time course of the biodistribution of boron was examined. At low injected doses normally used (5–10 mg ¹⁰B/kg), a peak tumor boron concentration of 35 μg of boron per g of tumor and a tumor/blood boron ratio of ~8 was achieved. The incorporation of both MAC and the hydrophilic species, TAC (Na₃[ae-B₂₀H₁₇NH₃]), within the same liposomes significantly enhanced biodistribution characteristics as exemplified by the maximum tumor boron concentration of 50 μg of boron per g of tumor and the tumor/blood boron ratio of 6. This MAC-TAC liposome was administered i.v. in oral cancer model hamsters. After 48 h, the boron concentration in tumors was 67 ppm whereas the precancerous tissue contained 11 ppm. Neutron irradiation resulted in an overall tumor response of 70% after 4 weeks. A high boron content in liposomes was achieved by encapsulating closo-dodecaborate derivatives, Na₂[B₁₂H₁₂], Na[B₁₂H₁₁NH₃], and BSH. The use of spermidin ium (spd) 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. Maximum boron concentrations of 202.7 and 82.4 ppm in tumor were achieved 36 h after injection at doses of 100 and 30 mg[B]/kg, respectively. Furthermore, 100% and 70% survivals were observed in tumor-bearing mice treated with spd-[BSH]-encapsulating liposomes at doses of 30 and 15 mg [B]/kg, respectively, 100 days after BNCT. Transferrin (TF) -receptor-mediated endocytosis is a normal physiological process by which TF delivers iron to cells, and a high concentration of TF receptor has been observed on most tumor cells in comparison with normal cells. Therefore, BSH-encapsulating TF-conjugated PEGylated liposomes (TF-PEG liposomes) were developed and shown to maintain a high ¹⁰B level in the tumor, with concentrations exceeding 30 μg of boron per g of tumor for at least 72 h after injection. On the other hand, plasma ¹⁰B level decreased, resulting in a tumor/plasma ratio of 6.0 at 72 h after injection. The administration of BSH encapsulated in TF-PEG liposomes at a dose of 5 or 20 mg ¹⁰B/kg followed by neutron irradiation suppressed tumor growth and improved long-term survival compared with PEG liposomes, bare liposomes, and free BSH. The biodistribution of BSH- and Na₂[B₁₀H₁₀]-encapsulated TF-PEG liposomes in SCC VII tumor-bearing mice was evaluated. The time courses of the change in ¹⁰B concentration in the tumors loaded with either of the two liposomes were similar, except that ¹⁰B concentrations were higher at 24 h for Na₂[B₁₀H₁₀]- than BSH-encapsulated TF-PEG liposomes, and ¹⁰B concentration in the tumors was 35.6 μg of boron per g of tumor when Na₂[B₁₀H₁₀]-encapsulated TF-PEG liposomes (35 mg ¹⁰B/kg) were injected.