Bioorganic & Medicinal Chemistry Letters 25 (2015) 172–174
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Antitumor effect of boron nitride nanotubes in combination with thermal neutron irradiation on BNCT Hiroyuki Nakamura a,b,⇑, Hayato Koganei b, Tatsuro Miyoshi b, Yoshinori Sakurai c, Koji Ono c, Minoru Suzuki c a b c
Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan Department of Chemistry, Faculty of Science, Gakushuin University, Mejiro, Toshima-ku, Tokyo 171-8588, Japan Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka 590-0494, Japan
a r t i c l e
i n f o
Article history: Received 8 September 2014 Revised 30 November 2014 Accepted 3 December 2014 Available online 8 December 2014 Keywords: Boron neutron capture therapy (BNCT) Boron nitride nanotubes (BNNTs) Antitumor effect B16 melanoma cells Colony-forming assay
a b s t r a c t The first BNCT antitumor effects of BNNTs toward B16 melanoma cells were demonstrated. The use of DSPE-PEG2000 was effective for preparation of the BNNT-suspended aqueous solution. BNNT–DSPEPEG2000 accumulated in B16 melanoma cells approximately three times higher than BSH and the higher BNCT antitumor effect was observed in the cells treated with BNNT–DSPE-PEG2000 compared to those treated with BSH, indicating that BNNT–DSPE-PEG2000 would be a possible candidate as a boron delivery vehicle for BNCT. Ó 2014 Elsevier Ltd. All rights reserved.
Boron neutron capture therapy (BNCT) for the treatment of cancer gained intense interest in recent years.1–3 This therapy is based on nuclear reaction of essentially nontoxic 10B species and low-energy thermal neutrons (0.025 eV), that yields high linear energy transfer (LET) particles, 7Li nuclei and 4He (a-particles), bearing energy of approximately 2.4 MeV (Eq. 1). These particles travel a distance equivalent to one cell diameter (approximately 5–9 lm) to destroy cells. The key for successful BNCT highly depends on the selective and efficient boron delivery. For this purpose, various boron carriers have been studied so far.4–8 10
B þ 1 n ! 4 HeðaÞ þ 7 Li þ 2:4 MeV
ð1Þ
Carbon nanotubes (CNTs), first discovered by Ijima in 1991,9 have been attracting growing interest due to their unique properties including peculiar shape, nanoscaled size, high thermal stability, and excellent conductivity. These superb mechanical and electronic properties have made them very promising new applications in chemistry and physics, particularly for the development of new nanotechnologies.10 CNTs have also been studied for application as a drug delivery carrier in biological systems.11–13 Especially, the functionalized CNTs with a range of 100–300 nm length escape from uptake by the reticuloendothelial system, revealing longer blood circulation times14 (Fig. 1). ⇑ Corresponding author. Tel.: +81 45 924 5244; fax: +81 45 924 5976. E-mail address: [email protected] (H. Nakamura). http://dx.doi.org/10.1016/j.bmcl.2014.12.005 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Like CNTs, boron nitride nanotubes (BNNTs) are currently attracting wide attention in the scientific community. BNNTs were first discovered in 1995.15 BNNTs are structural analogues of CNTs, in which carbon atoms are fully substituted by alternating boron and nitrogen atoms, and possess advantages over carbon nanotubes: electrical insulating property and thermal and chemical stability.16–18 Furthermore, BNNTs are lightweight, have excellent mechanical properties, a stronger resistance to oxidation than carbon nanotubes, and biocompatibility, offering a multifunctional material in nanocomposite materials,19 nanoscale electrical devices,20 biomedical fields,21 optical systems,19 and promising space radiation shielding applications.22 Meanwhile, BNNTs have been considered to be possible candidates as a boron agent for BNCT because of their high boron density (ca. 50%) equivalents to hundreds to thousands per each nanotube.23–25 In this Letter, we demonstrated antitumor effect of BNNTs in combination with thermal neutron irradiation on cancer cells. We first conducted cytotoxicity experiments of BNNTs toward B16 melanoma cells and faced to the problem of their poor solubility in aqueous solutions. Several protocols have been reported for dispersion of BNNTs in water including polyethyleneimine- and poly-L-lysine-coating,26 assembly of glycodendrimers,21 non-covalent wrapping with glycol-chitosan27 and Tween 80 (polyoxyethylene sorbitan monooleate),28 and functionalization with methoxy-poly(ethyleneglycol)-1,2-distearoyl-sn-glycero-3phosphoethanolamine (mPEG-DSPE; average molecular weight of
H. Nakamura et al. / Bioorg. Med. Chem. Lett. 25 (2015) 172–174
173
Figure 1. Structures of carbon nanotubes (CNTs; left) and boron nitride nanotubes (BNNTs; right).
5000, Laysan Bio) conjugates29 on BNNT surface. Especially, the dispersion of the mPEG-DSPE functionalized BNNTs was reported to be stable for more than 3 months without noticeable aggregation. Therefore, we prepared the dispersed solution of BNNTs using the mPEG-DSPE protocol. DSPE-PEG2000 (SUNBRIGHT DSPE-020CN, NOF Co.) was chosen and effect of amounts of DSPE-PEG2000 on dispersion of BNNTs was first examined. The dispersion levels were evaluated by measuring the boron concentration of the supernatant BNNTs solution obtained after sonication for 2 h followed by centrifugation for 1 h with 3000 rpm. The results are shown in Figure 2. Boron concentrations in the BNNT solutions increased with increasing DSPE-PEG2000 concentrations. At 600 lM concentration of DSPE-PEG2000, the BNNT solution became saturated and the boron concentrations dropped at higher DSPE-PEG2000 concentrations. These results indicate that the condition of 73.6 ppm B/600 lM DSPE-PEG2000 is the best for preparation of the BNNT-dispersing aqueous solution. The particle size distribution and zeta potential were 872 ± 147 nm and 35.3 ± 14.8 mV, respectively, measured with an electrophoretic light scattering spectrophotometer (Nano-ZS, Sysmex, Japan). As shown in Figure 3, well-dispersed BNNTs in water were observed in the presence of DSPE-PEG2000. We next examined cytotoxicity of BNNTs using the dispersed BNNT solution (BNNT–DSPE-PEG2000). B16 melanoma cells were incubated with various concentrations of BNNT–DSPE-PEG2000 (10–100 ppm boron concentrations) for 60 min. Incubation of cells in the presence of the dispersed BNNT solution containing a high DSPE-PEG2000 concentration resulted in dissolution of the cell membrane due to the detergent behavior of DSPE-PEG2000. After the medium was exchanged to the fresh, the cells were further
Figure 2. Effect of amounts of DSPE-PEG2000 on dispersion of BNNTs. A mixture of BNNTs (2 mg) and DSPE-PEG2000 in water (10 mL) was sonicated for 2 h at room temperature and centrifugation of the resulting suspended solution was carried out at 4 °C for 1 h with 3000 rpm. Boron concentration of the supernatant was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES, HORIBA, Japan).
Figure 3. Dispersion of BNNTs in water. BNNTs in water after 2 h of sonication in the absence (left) or in the presence (right) of DSPE-PEG2000. Well-dispersed BNNTs in water were observed in the right glass tube.
incubated in the BNNT–DSPE-PEG2000 free medium for 72 h and then the cell viability was determined by the MTT assay. As shown in Figure 4, significant cytotoxicity of BNNTs toward B16 cells was not observed at up to 100 ppm boron concentration of the dispersed BNNTs in the cell medium. Therefore, we examined boron accumulation in B16 melanoma cells at various concentrations of BNNT–DSPE-PEG2000 (10–100 ppm B). We used sodium mercaptoundecahydrododecaborate (BSH), which has been used for a clinical treatment of glioblastoma patients in BNCT as a control. B16 melanoma cells (1 106 cells) were incubated at 37 °C for 1 h in a medium containing BNNT–DSPE-PEG2000. After the incubation, the cells were washed three times with PBS and digested with 2 mL of perchloric acid/hydrogen peroxide at 70 °C for 1 h. Boron concentrations in the solution were determined by an inductively coupled plasma atomic emission spectroscopy (ICP-AES). The results are shown in Figure 5. Boron concentration in the B16 cells increased in a dose-dependent manner and a boron concentration of 0.22 lg/106 cells was observed at a 100 ppm B concentration of BNNT–DSPE-PEG2000. In the case of BSH, approximately 0.2 lg/106 cells was observed at a 300 ppm B concentration, indicating that BNNT–DSPE-PEG2000 accumulates in B16 cells approximately three times higher than BSH. However, L-BPA accumulated more effectively in B16 cells compared with BNNT–DSPE-PEG2000. It is known that L-BPA actively accumulates in many tumor cells through L-type amino acid transporter 1 (LAT1).30 Because the promising accumulation in B16 cells was observed, we further examined BNCT effects of BNNT–DSPE-PEG2000 on the survivals of B16 cells at the Heavy Water Column of the Kyoto
Figure 4. Effects of BNNTs on B16 melanoma cell viability. Cells were incubated with various concentrations of BNNTs (10–100 ppm boron concentrations) in a 96-well microplate at 37 °C in 5% CO2 in air for 60 min. After the medium was exchanged, the cells were further incubated in the absence of BNNTs for 72 h and then the cell viability was determined by the MTT assay.
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effects of both BNNT–DSPE-PEG2000 and BSH were not very strong due to their low boron 10 concentrations. In conclusion, we were the first to demonstrate the BNCT effects of BNNTs in B16 melanoma cells. The use of DSPE-PEG2000 was effective for preparation of the BNNT suspension in an aqueous solution. It was observed that BNNT–DSPE-PEG2000 accumulated in B16 cells approximately three times higher than BSH and that a higher BNCT antitumor effect was observed in the cells treated with BNNT–DSPE-PEG2000 compared to those treated with BSH, indicating that BNNT–DSPE-PEG2000 would be a possible candidate as a boron delivery vehicle for BNCT. Acknowledgments Figure 5. Boron accumulation in B16 melanoma cells at various concentrations of BNNT–DSPE-PEG2000 (10–100 ppm B). L-BPA (10 ppm) and BSH (300 ppm B) were used as controls.
We thank Professor Yoshio Bando at National Institute for Materials Science for his kind donation of BNNTs. This work was partially supported by the Ministry of Education, Science, Sports, Culture and Technology, Grant-in-Aid for Scientific Research (B) (No. 26293007) from Japan. References and notes
Figure 6. Clonogenic cell-survivals after irradiation in vitro using thermal neutron beams. BNNT–DSPE-PEG2000 (100 ppm natural B, d); BSH (100 ppm natural B, N); control (}).
University Research Reactor (KUR). B-16 cells were seeded into 60 mm dishes in a compound-free medium. After pre-incubation for 24 h, the cells were incubated for 1 h in a medium containing BNNT–DSPE-PEG2000 or BSH and then trypsinized. The single-cell suspensions were placed into a Teflon tube and irradiated at room temperature by neutron beams at a power of 1 MW. The neutron fluence was measured from the radioactivation of gold foil. Contaminating gamma rays, including secondary gamma rays, were measured with thermoluminescence dosimeter (TLD) powder. The TLD used was beryllium oxide (BeO) enclosed in a quartz glass capsule. BeO itself is sensitive to thermal neutrons. The average neutron fluxes were 1.0 109 neutrons/cm2/s. Cell survival was defined using a colony-forming assay. The irradiated cells were seeded into 100 mm dishes at various densities depending on the physical dose that cells received, and cultured in a serum-containing medium. After 14 d, the colonies were stained with methylene blue. A cell cluster containing at least 50 cells was considered a single colony. The surviving fraction was calculated as the number of colonies of treated cells divided by that for the control cells. The neutron-dose dependent surviving fractions are shown in Figure 6. BNNT–DSPE-PEG2000 killed more effectively than BSH probably due to the higher boron accumulation in B16 cells indicated in Figure 4. Because 100 ppm of natural boron concentration was employed, boron 10 concentration in the medium was calculated to be 20 ppm. Therefore BNCT antitumor
1. Barth, R. F.; Coderre, J. A.; Vicente, M. G.; Blue, T. E. Clin. Cancer Res. 2005, 11, 3987. 2. Yamamoto, T.; Nakai, K.; Matsumura, A. Cancer Lett. 2008, 262, 143. 3. Soloway, A. H.; Tjarks, W.; Barnum, B. A.; Rong, F. G.; Barth, R. F.; Codogni, I. M.; Wilson, J. G. Chem. Rev. 1998, 98, 1515. 4. Yinghuai, Z.; Cheng Yan, K.; Maguire, J. A.; Hosmane, N. S. Curr. Chem. Biol. 2007, 1, 141. 5. Nakamura, H.; Kirihata, M. In Neutron Capture Therapy Principles and Applications; Sauerwein, W. A. G., Wittig, A., Moss, R., Nakagawa, Y., Eds.; Springer: Heidelberg, 2012; pp 99–116. 6. Calabrese, G.; Nesnas, J. J.; Barbu, E.; Fatouros, D.; Tsibouklis, J. Drug Discovery Today 2012, 17, 153. 7. Barth, R. F.; Vicente, M. G.; Harling, O. K.; Kiger, W. S.; Riley, K. J.; Binns, P. J.; Wagner, F. M.; Suzuki, M.; Aihara, T.; Kato, I.; Kawabata, S. Radiat. Oncol. 2012, 7, 146. 8. Moss, R. L. Appl. Radiat. Isot. 2014, 88, 2. 9. Iijima, S. Nature 1991, 354, 56. 10. De Volder, M. F. L.; Tawfick, S. H.; Baughman, R. H.; Hart, A. J. Science 2013, 339, 535. 11. Pantarotto, D.; Briand, J. P.; Prato, M.; Bianco, A. Chem. Commun. 2004, 16. 12. Kam, N. W. S.; O’Connell, M.; Wisdom, J. A.; Dai, H. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11600. 13. Yinghuai, Z.; Peng, A. T.; Carpenter, K.; Maguire, J. A.; Hosmane, N. S.; Takagaki, M. J. Am. Chem. Soc. 2005, 127, 9875. 14. Liu, Z.; Cai, W. B.; He, L. N.; Nakayama, N.; Chen, K.; Sun, X. M.; Chen, X. Y.; Dai, H. J. Nat. Nanotechnol. 2007, 2, 47. 15. Chopra, N. G.; Luyken, R. J.; Cherrey, K.; Crespi, V. H.; Cohen, M. L.; Louie, S. G.; Zettl, A. Science 1995, 269, 966. 16. Han, W. Q.; Mickelson, W.; Cumings, J.; Zettl, A. Appl. Phys. Lett. 2002, 81, 1110. 17. Golberg, D.; Bando, Y.; Tang, C. C.; Zhi, C. Y. Adv. Mater. 2007, 19, 2413. 18. Golberg, D.; Bando, Y.; Huang, Y.; Terao, T.; Mitome, M.; Tang, C. C.; Zhi, C. Y. ACS Nano 2010, 4, 2979. 19. Zhi, C.; Bando, Y.; Tang, C.; Xie, R.; Sekiguchi, T.; Golberg, D. J. Am. Chem. Soc. 2005, 127, 15996. 20. Wang, W.; Bando, Y.; Zhi, C.; Fu, W.; Wang, E.; Golberg, D. J. Am. Chem. Soc. 2008, 130, 8144. 21. Chen, X.; Wu, P.; Rousseas, M.; Okawa, D.; Gartner, Z.; Zettl, A.; Bertozzi, C. R. J. Am. Chem. Soc. 2009, 131, 890. 22. Yu, J.; Chen, Y.; Elliman, R. G.; Petravic, M. Adv. Mater. 2006, 18, 2157. 23. Menichetti, L.; De Marchi, D.; Calucci, L.; Ciofani, G.; Menciassi, A.; Forte, C. Appl. Radiat. Isot. 2011, 69, 1725. 24. Ciofani, G.; Raffa, V.; Menciassi, A.; Cuschieri, A. Nanoscale Res. Lett. 2009, 4, 113. 25. Yinghuai, Z.; Hosmane, N. S. Future Med. Chem. 2013, 5, 705. 26. Ciofani, G.; Raffa, V.; Menciassi, A.; Dario, P. J. Nanosci. Nanotechnol. 2008, 8, 6223. 27. Ciofani, G.; Danti, S.; D’Alessandro, D.; Moscato, S.; Menciassi, A. Biochem. Biophys. Res. Commun. 2010, 394, 405. 28. Horváth, L.; Magrez, A.; Golberg, D.; Zhi, C.; Bando, Y.; Smajda, R.; Horváth, E.; Forró, L.; Schwaller, B. ACS Nano 2011, 5, 3800. 29. Lee, C. H.; Zhang, D.; Yap, Y. K. J. Phys. Chem. C 2011, 116, 1798. 30. Wittig, A.; Sauerwein, W. A.; Coderre, J. A. Radiat. Res. 2000, 153, 173.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Antitumor effect of boron nitride nanotubes in combination with thermal neutron irradiation on BNCT Hiroyuki Nakamura a,b,⇑, Hayato Koganei b, Tatsuro Miyoshi b, Yoshinori Sakurai c, Koji Ono c, Minoru Suzuki c a b c
Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta-cho, Midori-ku, Yokohama 226-8503, Japan Department of Chemistry, Faculty of Science, Gakushuin University, Mejiro, Toshima-ku, Tokyo 171-8588, Japan Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka 590-0494, Japan
a r t i c l e
i n f o
Article history: Received 8 September 2014 Revised 30 November 2014 Accepted 3 December 2014 Available online 8 December 2014 Keywords: Boron neutron capture therapy (BNCT) Boron nitride nanotubes (BNNTs) Antitumor effect B16 melanoma cells Colony-forming assay
a b s t r a c t The first BNCT antitumor effects of BNNTs toward B16 melanoma cells were demonstrated. The use of DSPE-PEG2000 was effective for preparation of the BNNT-suspended aqueous solution. BNNT–DSPEPEG2000 accumulated in B16 melanoma cells approximately three times higher than BSH and the higher BNCT antitumor effect was observed in the cells treated with BNNT–DSPE-PEG2000 compared to those treated with BSH, indicating that BNNT–DSPE-PEG2000 would be a possible candidate as a boron delivery vehicle for BNCT. Ó 2014 Elsevier Ltd. All rights reserved.
Boron neutron capture therapy (BNCT) for the treatment of cancer gained intense interest in recent years.1–3 This therapy is based on nuclear reaction of essentially nontoxic 10B species and low-energy thermal neutrons (0.025 eV), that yields high linear energy transfer (LET) particles, 7Li nuclei and 4He (a-particles), bearing energy of approximately 2.4 MeV (Eq. 1). These particles travel a distance equivalent to one cell diameter (approximately 5–9 lm) to destroy cells. The key for successful BNCT highly depends on the selective and efficient boron delivery. For this purpose, various boron carriers have been studied so far.4–8 10
B þ 1 n ! 4 HeðaÞ þ 7 Li þ 2:4 MeV
ð1Þ
Carbon nanotubes (CNTs), first discovered by Ijima in 1991,9 have been attracting growing interest due to their unique properties including peculiar shape, nanoscaled size, high thermal stability, and excellent conductivity. These superb mechanical and electronic properties have made them very promising new applications in chemistry and physics, particularly for the development of new nanotechnologies.10 CNTs have also been studied for application as a drug delivery carrier in biological systems.11–13 Especially, the functionalized CNTs with a range of 100–300 nm length escape from uptake by the reticuloendothelial system, revealing longer blood circulation times14 (Fig. 1). ⇑ Corresponding author. Tel.: +81 45 924 5244; fax: +81 45 924 5976. E-mail address: [email protected] (H. Nakamura). http://dx.doi.org/10.1016/j.bmcl.2014.12.005 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Like CNTs, boron nitride nanotubes (BNNTs) are currently attracting wide attention in the scientific community. BNNTs were first discovered in 1995.15 BNNTs are structural analogues of CNTs, in which carbon atoms are fully substituted by alternating boron and nitrogen atoms, and possess advantages over carbon nanotubes: electrical insulating property and thermal and chemical stability.16–18 Furthermore, BNNTs are lightweight, have excellent mechanical properties, a stronger resistance to oxidation than carbon nanotubes, and biocompatibility, offering a multifunctional material in nanocomposite materials,19 nanoscale electrical devices,20 biomedical fields,21 optical systems,19 and promising space radiation shielding applications.22 Meanwhile, BNNTs have been considered to be possible candidates as a boron agent for BNCT because of their high boron density (ca. 50%) equivalents to hundreds to thousands per each nanotube.23–25 In this Letter, we demonstrated antitumor effect of BNNTs in combination with thermal neutron irradiation on cancer cells. We first conducted cytotoxicity experiments of BNNTs toward B16 melanoma cells and faced to the problem of their poor solubility in aqueous solutions. Several protocols have been reported for dispersion of BNNTs in water including polyethyleneimine- and poly-L-lysine-coating,26 assembly of glycodendrimers,21 non-covalent wrapping with glycol-chitosan27 and Tween 80 (polyoxyethylene sorbitan monooleate),28 and functionalization with methoxy-poly(ethyleneglycol)-1,2-distearoyl-sn-glycero-3phosphoethanolamine (mPEG-DSPE; average molecular weight of
H. Nakamura et al. / Bioorg. Med. Chem. Lett. 25 (2015) 172–174
173
Figure 1. Structures of carbon nanotubes (CNTs; left) and boron nitride nanotubes (BNNTs; right).
5000, Laysan Bio) conjugates29 on BNNT surface. Especially, the dispersion of the mPEG-DSPE functionalized BNNTs was reported to be stable for more than 3 months without noticeable aggregation. Therefore, we prepared the dispersed solution of BNNTs using the mPEG-DSPE protocol. DSPE-PEG2000 (SUNBRIGHT DSPE-020CN, NOF Co.) was chosen and effect of amounts of DSPE-PEG2000 on dispersion of BNNTs was first examined. The dispersion levels were evaluated by measuring the boron concentration of the supernatant BNNTs solution obtained after sonication for 2 h followed by centrifugation for 1 h with 3000 rpm. The results are shown in Figure 2. Boron concentrations in the BNNT solutions increased with increasing DSPE-PEG2000 concentrations. At 600 lM concentration of DSPE-PEG2000, the BNNT solution became saturated and the boron concentrations dropped at higher DSPE-PEG2000 concentrations. These results indicate that the condition of 73.6 ppm B/600 lM DSPE-PEG2000 is the best for preparation of the BNNT-dispersing aqueous solution. The particle size distribution and zeta potential were 872 ± 147 nm and 35.3 ± 14.8 mV, respectively, measured with an electrophoretic light scattering spectrophotometer (Nano-ZS, Sysmex, Japan). As shown in Figure 3, well-dispersed BNNTs in water were observed in the presence of DSPE-PEG2000. We next examined cytotoxicity of BNNTs using the dispersed BNNT solution (BNNT–DSPE-PEG2000). B16 melanoma cells were incubated with various concentrations of BNNT–DSPE-PEG2000 (10–100 ppm boron concentrations) for 60 min. Incubation of cells in the presence of the dispersed BNNT solution containing a high DSPE-PEG2000 concentration resulted in dissolution of the cell membrane due to the detergent behavior of DSPE-PEG2000. After the medium was exchanged to the fresh, the cells were further
Figure 2. Effect of amounts of DSPE-PEG2000 on dispersion of BNNTs. A mixture of BNNTs (2 mg) and DSPE-PEG2000 in water (10 mL) was sonicated for 2 h at room temperature and centrifugation of the resulting suspended solution was carried out at 4 °C for 1 h with 3000 rpm. Boron concentration of the supernatant was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES, HORIBA, Japan).
Figure 3. Dispersion of BNNTs in water. BNNTs in water after 2 h of sonication in the absence (left) or in the presence (right) of DSPE-PEG2000. Well-dispersed BNNTs in water were observed in the right glass tube.
incubated in the BNNT–DSPE-PEG2000 free medium for 72 h and then the cell viability was determined by the MTT assay. As shown in Figure 4, significant cytotoxicity of BNNTs toward B16 cells was not observed at up to 100 ppm boron concentration of the dispersed BNNTs in the cell medium. Therefore, we examined boron accumulation in B16 melanoma cells at various concentrations of BNNT–DSPE-PEG2000 (10–100 ppm B). We used sodium mercaptoundecahydrododecaborate (BSH), which has been used for a clinical treatment of glioblastoma patients in BNCT as a control. B16 melanoma cells (1 106 cells) were incubated at 37 °C for 1 h in a medium containing BNNT–DSPE-PEG2000. After the incubation, the cells were washed three times with PBS and digested with 2 mL of perchloric acid/hydrogen peroxide at 70 °C for 1 h. Boron concentrations in the solution were determined by an inductively coupled plasma atomic emission spectroscopy (ICP-AES). The results are shown in Figure 5. Boron concentration in the B16 cells increased in a dose-dependent manner and a boron concentration of 0.22 lg/106 cells was observed at a 100 ppm B concentration of BNNT–DSPE-PEG2000. In the case of BSH, approximately 0.2 lg/106 cells was observed at a 300 ppm B concentration, indicating that BNNT–DSPE-PEG2000 accumulates in B16 cells approximately three times higher than BSH. However, L-BPA accumulated more effectively in B16 cells compared with BNNT–DSPE-PEG2000. It is known that L-BPA actively accumulates in many tumor cells through L-type amino acid transporter 1 (LAT1).30 Because the promising accumulation in B16 cells was observed, we further examined BNCT effects of BNNT–DSPE-PEG2000 on the survivals of B16 cells at the Heavy Water Column of the Kyoto
Figure 4. Effects of BNNTs on B16 melanoma cell viability. Cells were incubated with various concentrations of BNNTs (10–100 ppm boron concentrations) in a 96-well microplate at 37 °C in 5% CO2 in air for 60 min. After the medium was exchanged, the cells were further incubated in the absence of BNNTs for 72 h and then the cell viability was determined by the MTT assay.
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effects of both BNNT–DSPE-PEG2000 and BSH were not very strong due to their low boron 10 concentrations. In conclusion, we were the first to demonstrate the BNCT effects of BNNTs in B16 melanoma cells. The use of DSPE-PEG2000 was effective for preparation of the BNNT suspension in an aqueous solution. It was observed that BNNT–DSPE-PEG2000 accumulated in B16 cells approximately three times higher than BSH and that a higher BNCT antitumor effect was observed in the cells treated with BNNT–DSPE-PEG2000 compared to those treated with BSH, indicating that BNNT–DSPE-PEG2000 would be a possible candidate as a boron delivery vehicle for BNCT. Acknowledgments Figure 5. Boron accumulation in B16 melanoma cells at various concentrations of BNNT–DSPE-PEG2000 (10–100 ppm B). L-BPA (10 ppm) and BSH (300 ppm B) were used as controls.
We thank Professor Yoshio Bando at National Institute for Materials Science for his kind donation of BNNTs. This work was partially supported by the Ministry of Education, Science, Sports, Culture and Technology, Grant-in-Aid for Scientific Research (B) (No. 26293007) from Japan. References and notes
Figure 6. Clonogenic cell-survivals after irradiation in vitro using thermal neutron beams. BNNT–DSPE-PEG2000 (100 ppm natural B, d); BSH (100 ppm natural B, N); control (}).
University Research Reactor (KUR). B-16 cells were seeded into 60 mm dishes in a compound-free medium. After pre-incubation for 24 h, the cells were incubated for 1 h in a medium containing BNNT–DSPE-PEG2000 or BSH and then trypsinized. The single-cell suspensions were placed into a Teflon tube and irradiated at room temperature by neutron beams at a power of 1 MW. The neutron fluence was measured from the radioactivation of gold foil. Contaminating gamma rays, including secondary gamma rays, were measured with thermoluminescence dosimeter (TLD) powder. The TLD used was beryllium oxide (BeO) enclosed in a quartz glass capsule. BeO itself is sensitive to thermal neutrons. The average neutron fluxes were 1.0 109 neutrons/cm2/s. Cell survival was defined using a colony-forming assay. The irradiated cells were seeded into 100 mm dishes at various densities depending on the physical dose that cells received, and cultured in a serum-containing medium. After 14 d, the colonies were stained with methylene blue. A cell cluster containing at least 50 cells was considered a single colony. The surviving fraction was calculated as the number of colonies of treated cells divided by that for the control cells. The neutron-dose dependent surviving fractions are shown in Figure 6. BNNT–DSPE-PEG2000 killed more effectively than BSH probably due to the higher boron accumulation in B16 cells indicated in Figure 4. Because 100 ppm of natural boron concentration was employed, boron 10 concentration in the medium was calculated to be 20 ppm. Therefore BNCT antitumor
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