Bioconjugate Chem. 2006, 17, 1314−1320
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Transferrin-Loaded nido-Carborane Liposomes: Tumor-Targeting Boron Delivery System for Neutron Capture Therapy Yusuke Miyajima,† Hiroyuki Nakamura,*,† Yasuhiro Kuwata,‡ Jong-Dae Lee,† Shinichiro Masunaga,§ Koji Ono,§ and Kazuo Maruyama*,‡ Department of Chemistry, Faculty of Science, Gakushuin University, Tokyo 171-8588, Department of Biopharmaceutics, School of Pharmaceutical Science, Teikyo University, Kanagawa 199-0195, and Research Reactor Institute, Kyoto University, Osaka 590-0494, Japan. Received March 13, 2006; Revised Manuscript Received July 8, 2006
The nido-carborane lipid 2 as a double-tailed boron lipid was synthesized from heptadecanol in five steps. The lipid 2 formed stable liposomes at 25% molar ratio toward DSPC with cholesterol. Transferrin was able to be introduced on the surface of boron liposomes (Tf(+)-PEG-CL liposomes) by the coupling of transferrin to the PEG-CO2H moieties of Tf(-)-PEG-CL liposomes. The biodistribution of Tf(+)-PEG-CL liposomes, in which 125I-tyraminyl inulins were encapsulated, showed that Tf(+)-PEG-CL liposomes accumulated in tumor tissues and stayed there for a sufficiently long time to increase tumor/blood concentration ratio, although Tf(-)-PEGCL liposomes were gradually released from tumor tissues with time. A boron concentration of 22 ppm in tumor tissues was achieved by the injection of Tf(+)-PEG-CL liposomes at 7.2 mg/kg body weight boron in tumorbearing mice. After neutron irradiation, the average survival rate of mice not treated with Tf(+)-PEG-CL liposomes was 21 days, whereas that of the treated mice was 31 days. Longer survival rates were observed in the mice treated with Tf(+)-PEG-CL liposomes; one of them even survived for 52 days after BNCT.
INTRODUCTION Boron neutron capture therapy (BNCT) was first proposed as a binary approach to cancer treatment in 1936 (1). This therapy is based on the capture reaction of thermal neutrons using nonradioactive 10B, which produces an R-particle and a lithium-7 nuclei ion with approximately 2.4 MeV. These highlinear-energy transfer particles dissipate their kinetic energy before traveling one cell diameter (5-9 µm) in biological tissues, ensuring their potential for precise cell killing (2-4). Their destructive effect is highly observed in boron-loaded tissues. Therefore, the successful treatment of cancer by BNCT demands the selective and marked accumulation of 10B in malignant tumor tissues. The amount of 10B required to obtain fatal tumor cell damage has been calculated to be more than 30 µg/g of tumor tissue, owing to the low contact probability between thermal neutrons and 10B (5). Therefore, the marked accumulation and selective delivery of boron into tumor tissues are the most important requirements to achieve an effective BNCT of cancers. Although mercaptoundecahydrododecaborate (BSH) (6, 7) and L-4-dihydroxyboronylphenylalanine (L-BPA) (8, 9) have been utilized for BNCT, new boron-10 carriers that deliver an adequate concentration of 10B atoms to tumors should be developed to achieve a potent and extensive cancer therapy (10). Recent promising approaches that meet this requirement entail the use of small boron molecules (2) and boron-conjugated biological complexes, such as monoclonal antibodies (11-17), epidermal growth factors (18-21), and carborane oligomers (22-26). We have developed a new type of target-sensitive liposomebearing polyethylene glycol (PEG), in which antibodies or * To whom correspondence should be addressed. Phone: +81-33986-0221. Fax: +81-3-5992-1029. E-mail: hiroyuki.nakamura@ gakushuin.ac.jp. † Gakushuin University. ‡ Teikyo University. § Kyoto University.
specific ligands are coupled with PEG chains (27-33). PEG units have a heightened effect of a prolonged residence time in the circulation and escaping ability from reticulo-endothelialsystem (RES) uptake (34, 35), resulting in an enhanced vascular permeability in solid tumor tissues (36). Furthermore, transferrin (Tf)-loaded PEG liposomes are internalized into tumor cells by receptor-mediated endocytosis and taken up into endosome-like intracellular vesicles (37). This phenomenon is due to a higher Tf receptor concentration in tumor cells than that in normal cells (38-40). We have applied the selective delivery by and cellentry mechanism of Tf-PEG liposomes in the boron delivery system for BNCT. In this system, boron compounds are encapsulated in liposomes and delivered to tumor tissues. TfPEG liposomes encapsulating BSH achieve sufficient therapeutic 10B concentrations in solid tumor tissues in colon 26 tumorbearing mice (e.g., 35.5 µg/g in tumor tissue) and inhibit tumor cell growth (41). As an alternative boron delivery system, a system involving the accumulation of boron in the liposomal bilayer is highly potent, because drugs can be encapsulated into the vacant inner cell of a liposome. Therefore, boron and drugs may be simultaneously delivered to tumor tissues for BNCT and chemotherapy of cancer. Hawthorne and co-workers first introduced nido-carborane into the amphiphile 1 as a hydrophilic part (Figure 1) and examined liposomal boron delivery in mice using 1 and distearoylphosphatidylcholine (DSPC) (42, 43). We designed the nido-carborane lipid 2 (CL), which has a doubletailed moiety conjugated with nido-carborane as a hydrophilic function, and communicated the successful synthesis of 2 (Scheme 1) and the stable vesicle formation determined by transmission electron microscopy analysis (44). Here, we developed of transferrin-loaded nido-carborane PEG liposomes (Tf(+)-PEG-CL liposomes) from 2 and found that Tf(+)PEG-CL liposomes accumulate in tumor tissues for a prolonged time in tumor-bearing mice, thereby affecting the survival of such mice after neutron irradiation.
10.1021/bc060064k CCC: $33.50 © 2006 American Chemical Society Published on Web 08/10/2006
Transferrin-Loaded nido-Carborane Liposomes
Figure 1. Structures of nido-carborane lipids. Scheme 1 a
a Reagents: (a) (1) NaH, THF, (2) CH2dC(CH2Cl)2, 93%; (b) (1) BH3‚Me2S, (2) H2O2, NaOH, 71%; (c) (1) NaH, THF, (2) propargyl bromide, 58%; (d) B10H14, CH3CN, toluene, 80%; (e) NaOMe, MeOH, 57%.
MATERIALS AND METHODS General. 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, δ units), and coupling constant was expressed in units of hertz (Hz). IR spectra were measured on a Shimadzu FTIR-8200A spectrometer. Analytical thin-layer chromatography (TLC) was performed on 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. Human iron-saturated transferrin (Holo-Tf) was purchased from Sigma. (St. Louis, MO). Distearoylphosphatidylcholine (DSPC) (COATSOME MC-8080), DSPE-PEG-OMe (DSPE-020C), and DSPEPEG-O-(CH2)5CO2H (DSPE-034GC) were supplied by Nippon Oil and Fats (Tokyo, Japan). The average molecular weights of DSPE-020C and DSPE-034GC were 2136 and 3133, respectively, and their polydispersities were 1.03 and 1.04, respectively, as measured by gel permeation chromatography. 125I-Tyraminyl inulin was synthesized as described previously (30, 31). 3-Heptadecyloxy-2-heptadecyloxymethyl-1-propene (3). To a NaH (16.0 g, 0.34 mol) suspension in THF (100 mL) was added hexadecanol (26.9 g, 0.105 mol) in THF (100 mL) dropwise at 0 °C under Ar. The reaction temperature was increased to room temperature, and the reaction mixture was stirred for 4 h. The mixture was again cooled to 0 °C, and 2-chloromethyl-3-chloro-1-propene (5.5 mL, 47.6 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 h and then refluxed for 89 h. The reaction was quenched with saturated aqueous NH4Cl solution (50 mL), and the mixture was extracted with ether, washed with saturated aqueous NaCl solution, dried over anhydrous MgSO4, and then concentrated. Purification by silica gel column chromatography
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(hexane/ethyl acetate ) 50:1) gave 3 (25.1 g, 44.3 mmol, 93% yield) as a white solid: 1H NMR (CDCl3, 400 MHz) δ 5.14 (s, 2H), 3.94 (s, 4H), 3.38 (t, J ) 6.8 Hz, 4H), 1.55 (m, 4H), 1.23 (brs, 56H), 0.86 (t, J ) 7.2 Hz, 6H); 13C NMR (CDCl3, 100 MHz) δ 143.3, 113.3, 71.5, 70.6, 31.9, 29.7, 29.7, 29.6, 29.5, 29.4, 26.2, 22.7, 14.1; IR (KBr) 2916, 2848, 2804, 2731, 1666, 1473, 1380, 1109, 1078, 1045, 1010 cm-1; HRMS (ESI) m/z (M + Na)+ calcd for C38H76O2Na 587.5743, found 587.5795. 3-Heptadecyloxy-2-heptadecyloxymethylpropane-1-ol (4). To a solution of 3 (18.1 g, 31.9 mmol) in THF (300 mL) was added BH3‚Me2S (31.9 mmol) dropwise at 0 °C under Ar. The reaction temperature was increased to room temperature, and the reaction mixture was stirred for 6 h. The mixture was again cooled to 0 °C, and an aqueous NaOH solution (3 M, 100 mL) was added. The reaction mixture was stirred at 0 °C for 24 h, and then, H2O2 (30%, 100 mL) was added. After 12 h at room temperature with stirring, K2CO3 (5.0 g) was added, and the mixture was extracted with ether, washed with saturated aqueous NaCl solution, dried over anhydrous MgSO4, and then concentrated. The residue was passed through a short column chromatograph on SiO2 with hexane/ethyl acetate (30:1). Recrystallization from hexane gave 4 (13.3 g, 22.8 mmol, 71% yield) as a colorless crystal: 1H NMR (CDCl3, 400 MHz) δ 3.74 (t, J ) 5.2 Hz, 2H), 3.50 (m, 4H), 3.38 (t, J ) 5.2 Hz, 4H), 2.07 (quint, J ) 5.6 Hz, 1H), 1.53 (t, J ) 6.4 Hz, 4H), 1.23 (brs, 56H), 0.86 (t, J ) 6.8 Hz, 6H); 13C NMR (CDCl3, 100 MHz) δ 71.8, 71.2, 65.1, 41.4, 32.1, 29.9, 29.8, 29.7, 29.6, 26.3, 22.9, 14.3; IR (KBr) 3328, 2839, 2744, 1471, 1377, 1313, 1232, 1213, 1191, 1116, 1041 cm-1; HRMS (ESI) m/z (M + Na)+ calcd for C38H78O3Na 605.5848, found 605.5816. 3-Heptadecyloxy-2-heptadecyloxymethyl-1-propargyloxypropane (5). To a NaH (1.03 g, 25.7 mmol) suspension in THF (20 mL) was added 4 (3.0 g, 5.2 mmol) in THF (15 mL) dropwise at 0 °C under Ar. The temperature was increased to room temperature, and the reaction mixture was stirred for 3 h. The mixture was again cooled to 0 °C, and propargyl bromide (1.6 mL, 15.4 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 20 h. The reaction was quenched with saturated aqueous NH4Cl solution (20 mL), and the mixture was extracted with ether, washed with saturated aqueous NaCl solution, dried over anhydrous MgSO4, and then concentrated. Purification by silica gel column chromatography (hexane/ethyl acetate ) 30:1) gave 5 (1.87 g, 3.0 mmol, 58% yield) as a yellow solid: 1H NMR (CDCl3, 400 MHz) δ 4.41 (d, J ) 2.4 Hz, 2H), 3.55 (d, J ) 6.0 Hz, 2H), 3.43 (d, J ) 6.4 Hz, 4H), 3.38 (t, J ) 6.4 Hz, 4H), 2.39 (s, 1H), 2.14 (m, 1H), 1.52 (t, J ) 6.8 Hz, 4H), 1.24 (brs, 56H), 0.87 (t, J ) 6.8 Hz, 6H); 13C NMR (CDCl3, 100 MHz) δ 74.2, 71.4, 69.2, 69.0, 58.6, 40.4, 32.1, 29.9, 29.8, 29.7, 29.6, 26.4, 22.9, 14.3; IR (KBr) 3249, 2912, 2848, 2798, 1508, 1473, 1458, 1359, 1087 cm-1; HRMS (ESI) m/z (M + Na)+ calcd for C41H80O3Na 643.6005, found 643.5989. (3-Heptadecyloxy-2-heptadecyloxymethyl-1-propoxy)methylo-carborane (6). The mixture of 5 (0.61 g, 0.99 mmol), decaborane (0.15 g, 1.2 mmol), and acetonitrile (1.56 mL, 29.6 mmol) in toluene (20 mL) was stirred under reflux condition for 6 h. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (hexane/ethyl acetate ) 30:1) to give 6 (0.61 g, 0.83 mmol, 80% yield) as a white solid: 1H NMR (CDCl3, 400 MHz) δ 3.97 (brs, 1H), 3.51 (d, J ) 5.6 Hz, 2H), 3.40-1.40 (m, 10H), 2.09 (quint, J ) 6.0 Hz, 1H), 1.51 (t, J ) 6.8 Hz, 4H), 1.24 (brs, 56H), 0.86 (t, J ) 6.8 Hz, 6H); 13C NMR (CDCl3, 100 MHz) δ 72.3, 71.6, 70.4, 68.9, 57.7, 40.4, 32.1, 29.9, 29.8, 29.7, 29.6, 26.4, 22.9, 14.3; IR (KBr) 2918, 2850, 2572, 1465, 1377, 1112, 1095, 1012 cm-1; MS (MALDI) m/z (M + Na)+ C41H90O3B10Na 763.8.
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(3-Heptadecyloxy-2-heptadecyloxymethyl-1-propoxy)methylo-nido-carborane sodium salt (2). The mixture of 6 (0.92 g, 1.25 mmol) and NaOMe (0.68 g, 12.5 mmol) in MeOH (15 mL) was stirred under reflux condition for 14 h. The reaction mixture was diluted with water (20 mL), extracted with ethyl acetate, washed with saturated aqueous NaCl solution, dried over anhydrous MgSO4, and then concentrated. Purification by silica gel column chromatography (gradient from hexane/ethyl acetate ) 30:1 to ethyl acetate only) gave 2 (0.56 g, 0.72 mmol, 57% yield) as a white solid: 1H NMR (CDCl3, 400 MHz) δ 3.77(d, J ) 5.6 Hz, 2H), 3.52 (m, 6H), 3.40 (t, J ) 6.4 Hz, 4H), 2.09 (m, 1H), 1.54 (m, 4H), 1.24 (brs, 56H), 0.87 (t, J ) 6.8 Hz, 6H); 13C NMR (CDCl3, 100 MHz) δ 41.05, 32.31, 29.93, 29.87, 29.57, 26.02, 22.90, 14.32; IR (KBr) 3419, 3251, 2850, 2520, 1714, 1614, 1456, 1373 cm-1; MS (MALDI) m/z (M + Na)+ C41H89O3B9Na2 774.8. Preparation of Calcein Encapsulated CL and PEG-CL Liposomes. CL and PEG-CL liposomes were prepared from DSPC, CH, and CL (1:1:X ) 0-1, molar ratio) or DSPC, CH, DSPE-PEG-OMe, and CL (1:1:0.12:X ) 0-1, molar ratio) by the reverse-phase evaporation (REV) method (44). The representative procedure for the preparation of 25% CL containing the PEG-CL liposome is shown as follows: A mixture of DSPC (118 mg), CH (47 mg), DSPE-PEG-OMe (56 mg), and CL (29 mg) were dissolved in 2 mL of chloroform/diisopropylether mixture (1:1, v/v) in a round-bottom flask. An aqueous solution of calcein (100 mM, 2 mL) was added to the lipid solution to form 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, and then, the organic solvent was removed under vacuum in a rotary evaporator at 37 °C while being broken down repeatedly to obtain a suspension of liposomes. The liposomes obtained were subjected to extrusion 10 times through a polycarbonate membrane of 100-nm pore size, using an extruder device (Lipex Biomembrane, Canada) thermostated at 60 °C. Purification was accomplished by ultracentrifuging at 200 000g for 20 min at 4 °C (Hitachi CS120, S100AT5 rotor), and the pellets obtained were resuspended in PBS buffer. Liposome size was measured with an electrophoretic light scattering spectrophotometer (ELS-700, Otsuka Electroics, Tokyo). The size distribution of CL (25-100%) containing the PEG-CL liposome was indicated in Figure 1 in the Supporting Information. Stability of CL and PEG-CL Liposomes in Fetal Bovine Serum. The calcein-encapsulated CL and PEG-CL liposomes were added to a fetal bovine serum (FBS) (volume ratio of FBS/ vesicle solution ) 9:1), and the mixture was incubated at 37 °C with stirring. The fluorescence intensity of the FBS solution was measured at 0-24 h using an excitation wavelength of 490 nm with emission wavelength of 520 nm. The fluorescence intensity was also measured at each fraction after degradation of liposomes by the addition of a 5% aqueous solution of Triton X-100 to the FBS solution. Conjugation of Transferrin on Surface of Tf(-)-PEGCL Liposomes. Tf(-)-PEG-CL liposomes were prepared from DSPC, CH, DSPE-PEG-OMe, DSPE-PEG-O(CH2)5CO2H, and CL (molar ratio 1:1:0.11:0.021:0.25) according to the procedure described above. In the case of 125Ityraminyl inulin encapsulation, the Tf(-)-PEG-CL liposomes were prepared using an aqueous solution containing 125Ityraminyl inulin (500 µg lipid/200 µL), which was obtained according to the reported procedure (46). Tf(+)-PEG-CL liposomes were prepared by the coupling of transferrin to the PEG-CO2H moieties of Tf(-)-PEG-CL liposomes as previously reported (33). Briefly, to 1 mL of Tf(-)-PEG-CL liposomes (5.8 µmol lipid) in 1.25 mL Mes buffer (10 mM Mes/ 150 mM NaCl, pH 5.5), 21 µmol of EDC and 28 µmol of
Miyajima et al.
S-NHS were added, and the mixture was incubated for 15 min at room temperature. The mixture was loaded into a Sephadex G25 column equilibrated with Mes buffer, and liposome fractions were collected. Transferrin (1 mg) in a NaCl solution (150 mM) with PBS (final 250 µL, pH 7.4) was then added to the liposome solution, and the mixture was incubated for 3 h at room temperature with gentle stirring. Purification was accomplished by ultracentrifugation at 200 000g for 20 min at 4 °C, and the pellets obtained were resuspended in 2 mL of PBS. Tf(+)-PEG-CL liposomes obtained were converted into the diferric form by treatment with FeCl3-nitriloacetic acid solution (250 mM, 20 µL, pH 7.4). After the reaction, the suspension was purified by ultracentrifugation and resuspended in PBS. Biodistribution of Tf(+)-PEG-CL and Tf(-)-PEG-CL Liposomes in Tumor-Bearing Mice. Biodistribution studies were performed using male BALB/c mice (6 weeks old, 1618 g, Nihon SLC). Tumor-bearing mice were prepared by inoculating sc a suspension (5 × 106 cells) of colon 26 cells directly into their back. The mice were kept on regular mouse diet and water, and maintained under a standard light/dark cycle in an ambient atmosphere. These experiments were performed when the tumor was 7-9 mm in diameter. 125I-Tyraminyl inulin solution was encapsulated in the liposomes, and 100 µL of liposomes was injected into the mice (3 per group) via the tail vein. At selected time intervals after administration, the mice were lightly anesthetized, bled via the retro-orbital sinus, sacrificed by cervical dislocation, and dissected. Their organs were excised, and their 125I content was estimated by a liquid scintillation counter. BNCT for Tumor-Bearing Mice Using Tf(+)-PEG-CL Liposomes. Tumor-bearing mice were prepared by inoculating sc a suspension (5 × 106 cells) of colon 26 cells directly into their left thigh. Tf(+)-PEG-CL liposomes, which were prepared from the 10B-enriched nido-carborane lipid 2, was injected into colon 26 tumor-bearing mice (18-22 g; 8 per group) via a tail vein at a dose of 7.2 mg 10B/kg (720 ppm of 10B concentration; 200 µL of liposome solution). Seventy-two hours after iv injection, 4 mice in each group were sacrified by cervical dislocation and dissected. Organs were excised, and a 10B concentration in each organ was determined by prompt γ-ray spectroscopy. At the same time, the other 4 mice in each group were anesthetized with sodium pentobarbital solution and placed in an acrylic mouse holder (Iuchi, Tokyo), where their whole bodies except the tumor-implanted leg were shielded with acrylic resin. The mice were irradiated in the KUR atomic reactor for 37 min at a rate of 2 × 1012 neutrons/cm2. The antitumor effect of BNCT was evaluated on the basis of the survival of the mice. All protocol were approved by the Institutional Animal Care and Use Committee in Kyoto University.
RESULTS AND DISCUSSION Synthesis of Double-Tailed nido-Carborane Lipid 2. The synthetic route of nido-carborane lipid 2 is shown in Scheme 1. The reaction of 2 equiv of heptadecanol with 3-chloro-2chloromethyl-1-propene using NaH as base gave the diether 3 in 93% yield, and the hydroboration of 3 gave the corresponding alcohol 4 in 71% yield. The alcohol 4 was converted into the propargyl ether 5 in 48% yield by the treatment with propargyl bromide, and the decaborane coupling of 5 was carried out in the presence of acetonitrile in toluene under reflux condition to give the corresponding o-carborane 6 in 80% yield. The degradation of the carborane cage by the treatment with sodium methoxide in methanol afforded the nido-carborane lipid 2 (CL) in 57% yield. Stability of CL and PEG-CL Liposomes in FBS. PEG units conjugated on the surface of liposomes are important for a prolonged residence time in the circulation and escaping ability
Transferrin-Loaded nido-Carborane Liposomes
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Figure 2. Fluorescence intensities of calcein-encapsulated CL and PEG-CL liposomes in fetal bovine serum (FBS). The white plots show the fluorescence intensity of the FBS solution, and the black plots show that of the solution after destruction of liposomes by the addition of Triton X-100. The difference in fluorescence intensity between the black and white plots indicates calcein release from liposomes.
from RES uptake, promoting the extravasation of the liposomes into the solid tumor tissue on liposomal drug delivery system. Therefore, we first investigated the stability of calceinencapsulated CL and PEG unit-conjugated CL liposomes in FBS. To FBS was added a liposome solution (the volume ratio of FBS/liposome solution ) 9:1) and incubated at 37 °C with stirring. The fluorescence of the FBS solutions was measured at 0-24 h. The results are shown in Figure 2. Fluorescence intensity is plotted on the vertical axis, and incubation time is plotted on the horizontal axis. The white plots show the fluorescence intensity of the FBS solution containing liposomes, and the black plots show that of the solution after destruction of liposomes by the addition of Triton X-100. In the case of liposomes with 0% and 25% CL (Figure 2a,b), no increase in the fluorescence intensity of the FBS solutions was observed within 24 h. However, the fluorescence intensity of the liposome with 50% CL increased after 1 h of incubation at 37 °C with stirring (Figure 2c). A similar tendency was observed in the case of the PEG liposomes, and the fluorescence intensity of the PEG liposomes with 50% CL increased even after 5 min of incubation (Figure 2d-f). These results indicate that the liposomes containing 25% of CL were stable in the FBS solution at 37 °C at least for 24 h and that liposomes with higher than 50% CL lead to an unstable liposomal formation. The increased rate of fluorescence intensity after the treatment with Triton X-100 may be associated with the amounts of calceins encapsulated in liposomes. Comparison of the increased rates of fluorescence intensity between PEG-CL liposome solutions and CL liposome solutions after treatment with Triton X-100 shows that the latter is relatively higher (Figure 2a-c vs d-f). This means that the PEG functional group would lower the capacity of calcein encapsulation in liposomes. Therefore, the higher CL content of the liposomes led not only to instability in liposome formation but also suppressed calcein encapsulation.
Biodistribution of Tf(-)-PEG-CL and Tf(+)-PEG-CL Liposomes in Tumor-Bearing Mice. Liposomes, in which 125Ityraminyl inulins were encapsulated, were injected intravenously into colon 26 tumor-bearing mice. At selected time intervals after administration, the mice were lightly anesthetized, bled via the retro-orbital sinus, killed by cervical dislocation, and dissected. Their organs were excised, and their 125I content was estimated using a liquid scintillation counter. The time-dependent distributions of Tf(-)-PEG-CL and Tf(+)-PEG-CL liposomes in various tissues are shown in Figure 3. The rapid clearance of Tf(-)-PEG-CL and Tf(+)-PEG-CL liposomes was observed in the blood, lung, and kidney after 3 h of injection. In general, PEGylated liposomes possess a longer circulation time compared to nonstealth liposomes (34, 35). We examined the effect of CL on the circulation time of PEGylated liposomes and found that CL influenced a stealth property of the PEG-CL liposome (see Figure 4 in Supporting information). The enhanced accumulation of Tf(+)-PEG-CL liposomes in comparison with Tf(-)-PEG-CL liposome accumulation was observed in the spleen within 72 h. Tf(+)-PEG-CL liposomes accumulated in the liver and tumor gradually, whereas Tf(-)-PEG-CL liposomes were released from those organs 72 h after injection, although percentage doses of these liposomes were similar 24 h after injection. Surprisingly, more than 1.5% of the total dose injected accumulated in tumor tissues. This enhanced accumulation of Tf(+)-PEG-CL liposomes may reflect marked receptor-mediated endocytosis after binding to tumor cells (37). Since enhanced accumulation in tumor tissues and a high tumor/blood ratio were observed 72 h after the administration of Tf(+)-PEG-CL liposomes, we next investigated the 10B distribution and BNCT effect in tumorbearing mice. 10B Concentration in Various Organs 72 h after Injection of Tf-PEG-CL Liposome. 10B-enriched Tf(+)-PEG-CL liposomes were injected into tumor-bearing mice, in which colon
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Figure 3. Time course of biodistribution of Tf(-)-PEG-CL liposome (TF(-)) and the Tf(+)-PEG-CL liposome (TF(+)). Liposomes encapsulating 125 I-tyraminyl inulin (500 g lipid/200 µL) were injected into male BALB/c mice (7 weeks old, weighing 20-25 g) via the tail vein. The distribution of liposomes was measured by determining the radioactivity of each tissue. The percent dose/per gram in each tissue is plotted on the vertical axis, and the time (h) after administration is plotted on the horizontal axis.
Figure 4. 10B concentration in various tissues 72 h after injection of Tf(+)-PEG-CL liposomes into tumor-bearing mice. 10B-enriched Tf(+)-PEG-CL liposomes were injected into tumor-bearing mice, in which colon 26 cells were transplanted into the left thigh, via the tail vein at a dose of 7.2 mg 10B/kg (200 µL of liposome solution).
26 cells were transplanted into their left thigh, via the tail vein at a dose of 7.2 mg 10B/kg (200 µL of a liposome solution). Seventy-two hours after administration, 10B concentration in each organ was measured by prompt γ-ray spectroscopy. The results are shown in Figure 4. No boron accumulation was observed in the muscle, heart, and brain; however, the boron concentrations in the lung, kidney, and blood were approximately 10 ppm. Since no accumulation of Tf(+)-PEG-CL liposomes labeled with 125I-tyraminyl inulin was observed, as
shown in Figure 3, it is considered that the 10B concentrations detected in such organs may be due to the accumulation of the nido-carborane lipid 2, which was caused by the degradation of the parent liposomes. Enhanced accumulation of 10B was observed in the spleen and liver, and this does not conflict with the result of the biodistribution of Tf(+)-PEG-CL liposomes as shown in Figure 3. A high level of 10B concentration (22 ppm) in the tumor was observed in tumor tissues 72 h after the administration of Tf(+)-PEG-CL liposomes. Previously, we reported that BSH-encapsulated Tf(+)-PEG liposomes (40) accumulated in tumor tissues at 35.5 ppm 10B concentration 72 h after administration of 35 mg/kg body weight 10B. Therefore, 10B delivery to tumor tissues by Tf(+)-PEG-CL liposomes would be more efficient in comparison with that by BSHencapsulated Tf(+)-PEG liposomes. In both cases, the conjunction of transferrin to the surface of liposomes is essential for the enhanced and prolonged accumulation of 10B in tumor tissues. Survival of Tumor-Bearing Mice after BNCT. Besides the determination of 10B concentration in various organs, the mice were anesthetized with sodium pentobarbital solution 72 h after the administration of Tf(+)-PEG-CL liposomes and placed in an acrylic mouse holder, where their whole bodies, except their tumor-implanted leg, were shielded with acrylic resin. Neutron irradiation was carried out for 37 min at a rate of 2 × 1012 neutrons/cm2 in the KUR atomic reactor. The antitumor
Transferrin-Loaded nido-Carborane Liposomes
Figure 5. Survival curve of tumor-bearing mice after neutron irradiation for 37 min in KUR atomic reactor. The mice were injected with 7.2 mg 10B/kg of the Tf(+)-PEG-CL liposome and incubated for 72 h before irradiation. Control indicates survival rates of tumor-bearing mice after neutron irradiation without administration of Tf(+)-PEGCL liposomes.
effect of BNCT was evaluated on the basis of the survival of the mice, as shown in Figure 5. All the nontreated mice did not survive after 32 days of neutron irradiation, and their average survival rate was 21 days. Long survival rates were observed in the mice treated with Tf(+)-PEG-CL liposomes; one of them even survived for 52 days after neutron irradiation. The average survival rate of the treated mice was 31 days.
CONCLUSION We succeeded in the synthesis of the double-tailed boron lipid 2, which forms a stable liposome at 25% molar ratio toward DSPC with cholesterol. Transferrin was able to be conjugated to the surface of boron liposomes (Tf(+)-PEG-CL liposomes) by the coupling of transferrin to the PEG-CO2H moieties of PEG(-)-CL liposomes. A biodistribution experiment on Tf(+)-PEG-CL liposomes, in which 125I-tyraminyl inulins were encapsulated, showed that Tf(+)-PEG-CL liposomes accumulate in tumor tissues and stay there for a sufficiently long time to increase tumor/blood concentration ratio, although Tf(-)-PEG-CL liposomes were gradually released from tumor tissues with time. A 10B concentration of 22 ppm was achieved by injection with Tf(+)-PEG-CL liposomes at 7.2 mg 10B/ kg in tumor-bearing mice. Although no complete cure of the tumor was observed after neutron irradiation, prolonged survivals of the mice were observed. The current boron delivery system uses the accumulation of boron in the liposomal bilayer instead of in the inner cell of liposomes. It is noted that the inner cells of a liposome are still vacant; therefore, it is envisioned that drugs can be encapsulated into the Tf(+)PEG-CL liposome. In this system, boron and drugs can be simultaneously delivered to tumor tissues. The current investigation and liposomal delivery of the double-tailed boron lipid 2 possesses a dual-mode vehicle for BNCT and chemotherapy of cancer. Supporting Information Available: Size distributions of PEGCL liposomes, analysis of the conjugation of transferrin to liposomes, size distributions of Tf(-)-PEG-CL liposome and Tf(+)-PEG-CL liposome, and effect of CL on the circulation time and accumulation of PEG-conjugated liposomes in various tissues in mice. This material is available free of charge via the Internet at http://pubs.acs.org.
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