JP2008222585A - beta-1,3-GLUCAN/CARBORANE COMPLEX - Google Patents

beta-1,3-GLUCAN/CARBORANE COMPLEX Download PDF

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JP2008222585A
JP2008222585A JP2007059771A JP2007059771A JP2008222585A JP 2008222585 A JP2008222585 A JP 2008222585A JP 2007059771 A JP2007059771 A JP 2007059771A JP 2007059771 A JP2007059771 A JP 2007059771A JP 2008222585 A JP2008222585 A JP 2008222585A
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carborane
spg
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glucan
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JP5159125B2 (en
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Seiji Shinkai
征治 新海
Shingo Tamesue
真吾 為末
Ryoji Hirose
良治 広瀬
Kazuro Sakurai
和朗 櫻井
Takeshi Nagasaki
健 長崎
Sosuke Numata
宗典 沼田
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Japan Science and Technology Agency
Mitsui DM Sugar Co Ltd
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Mitsui Sugar Co Ltd
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a complex having water solubility and cell affinity for facilitating the introduction of carborane used for a boron-neutron capture therapy into a living body, and to provide a method for preparing the complex. <P>SOLUTION: The complex is obtained by allowing β-1,3-glucan of a natural polysaccharide to include the carborane. The method for preparing the complex includes a step for mixing the β-1,3-glucan and the carborane in a polar solvent solution, and maturing the product by adding water. Schizophyllan as the β-1,3-glucan and m-carborane as the carborane are preferably used. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

本発明はホウ素中性子捕捉療法に使用されるカルボランを、生体内へ導入しやすくするために水溶性かつ細胞親和性のある複合体に包接する技術に関する。   The present invention relates to a technique for encapsulating carborane used for boron neutron capture therapy in a water-soluble and cytophilic complex so that it can be easily introduced into a living body.

原子番号１０のホウ素のケージ状化合物であるカルボラン（図１参照）は新規ながん治療法であるホウ素中性子捕捉療法neutron capture therapy（BNCT）に用いる化合物として近年注目を集めている。ホウ素は同位体の存在比が大きく、そのため人体に無害な熱中性子線を照射すると核反応が生じ、この核反応によりホウ素はα線を照出する（図２参照）という興味深い性質を持つ。α線は飛程が9〜10 nm程度と極めて短く、この範囲の細胞のみを死滅させることができる。この性質を利用したホウ素中性子捕捉療法（BNCT）では、ガン細胞のまわりにホウ素を集積させ、ガン細胞のみを選択的に死滅させることを目的としている。カルボランは10 個ものホウ素を含む生物学的に安定なホウ素クラスターである。前記のごとくこの治療法の成否は、いかにしてガン細胞のみにカルボランを集積させるかにかかっている。しかしながら、カルボランは水に難溶で、細胞選択性もないため細胞に導入することや、ガン細胞の周りに集積させることは困難である。このためこれまでに細胞選択性や水溶性の向上、置換基修飾など様々な研究がなされている（非特許文献１-９）。
T. Takenobu, T.Takano, M. Shiraishi, Y. Murakami, M. Ata, H. Kataura, Y. Achiba, Y. Iwasa,Nature Materials, 2, 683 (2003) S. B. Kaul, R.A. Kaser, J. Am. Chem. Soc., 118, 1223 (1996). P. D. Godfrey,W. J. Crigsby, P. J. Nichols, C. L. Raston, J. Am. Chem. Soc., 119, 9283(1997). A. Harada, S. Takahashi, J. Chem. Commun.,12, 1352 (1988). M-J. Hardie,C-L. Raston., J. Chem. Commun., 12, 1153 (1999). M-J. Hardie,C-L. Raston., Eur. J. Inorg. Chem., 12, 195 (1999). L. Craciun, R.Custelcean, Inorg. Chem, 38, 4916 (1999). L. Deng, H-S.Chan, Z. Xie, J. Am. Chem. Soc, 128, 5219. (2006). A. H. Soloway,W. T. Beverly, A, Barnum, F-G. Rong, R-F. Barth, I-W. Codogni, J. G. Wilson,Chem. Rev., 98, 1515 (1998). In recent years, carborane (see FIG. 1), which is a cage compound of boron having an atomic number of 10, has attracted attention as a compound used for neutron capture therapy (BNCT), which is a novel cancer treatment. Boron has an abundance ratio of isotopes, and therefore, when a thermal neutron beam that is harmless to the human body is irradiated, a nuclear reaction occurs, and boron has an interesting property that it emits alpha rays (see FIG. 2). The α ray has an extremely short range of about 9 to 10 nm and can kill only cells in this range. Boron neutron capture therapy (BNCT) using this property aims to accumulate boron around cancer cells and selectively kill only cancer cells. Carborane is a biologically stable boron cluster containing as many as 10 boron. As described above, the success of this therapy depends on how carborane is accumulated only in cancer cells. However, since carborane is hardly soluble in water and does not have cell selectivity, it is difficult to introduce carborane into cells or to accumulate around cancer cells. For this reason, various studies have been made so far, such as improvement of cell selectivity, water solubility, and modification of substituents (Non-patent Documents 1-9).
T. Takenobu, T. Takano, M. Shiraishi, Y. Murakami, M. Ata, H. Kataura, Y. Achiba, Y. Iwasa, Nature Materials, 2, 683 (2003) SB Kaul, RA Kaser, J. Am. Chem. Soc., 118, 1223 (1996). PD Godfrey, WJ Crigsby, PJ Nichols, CL Raston, J. Am. Chem. Soc., 119, 9283 (1997). A. Harada, S. Takahashi, J. Chem. Commun., 12, 1352 (1988). MJ. Hardie, CL. Raston., J. Chem. Commun., 12, 1153 (1999). MJ. Hardie, CL. Raston., Eur. J. Inorg. Chem., 12, 195 (1999). L. Craciun, R. Custelcean, Inorg. Chem, 38, 4916 (1999). L. Deng, HS. Chan, Z. Xie, J. Am. Chem. Soc, 128, 5219. (2006). AH Soloway, WT Beverly, A, Barnum, FG. Rong, RF. Barth, IW. Codogni, JG Wilson, Chem. Rev., 98, 1515 (1998).

しかしながら、過去報告されている手法はカルボランの炭素部に種々の置換基を導入する手法がほとんどであり、思ったような機能の向上はなされていない。また、シクロデキストリンなどの包接化合物によるカルボランの複合化も報告されており、成功例としてβ-シクロデキストリンによる複合化があるが、β-シクロデキストリンは水溶性がα-およびγ-体に比べて悪いことが知られている。後にγ-シクロデキストリンによる包接も報告されているが、カルボラン自身に置換基を導入する必要があった。また、シクロデキストリンとの複合体を細胞に導入することは複合体の安定性などの問題も考えられる。   However, most of the methods reported in the past are methods of introducing various substituents into the carbon part of carborane, and the functions have not been improved as expected. In addition, carborane complexation with inclusion compounds such as cyclodextrin has been reported, and successful examples include complexation with β-cyclodextrin, but β-cyclodextrin is more soluble in water than α- and γ-forms. Is known to be bad. Although inclusion by γ-cyclodextrin was later reported, it was necessary to introduce a substituent into carborane itself. In addition, introduction of a complex with cyclodextrin into the cell may cause problems such as stability of the complex.

本発明の目的は、カルボランを生体に安全な媒体を用いることにより、水溶化および細胞親和性を向上させる技術を開発することにある。   An object of the present invention is to develop a technique for improving water solubilization and cell affinity by using carborane in a biologically safe medium.

本発明者は、天然の多糖であるシゾフィランに代表されるβ-1,3-グルカン内に、カルボン、特にm-カルボランを包接、内部空間に配列させることにより、カルボランを水溶化できることを見出し、本発明を導き出した。
かくして、本発明は、β-1,3-グルカンとカルボランから成る複合体を提供するものである。
さらに、本発明に従えば、上記の複合体を調製する方法であって、β-1,3-グルカンおよびカルボランを極性溶媒溶液中で混合し、水を加えて熟成する工程を含む方法が提供される。 The present inventor has found that carborane can be water-solubilized by inclusion of carvone, particularly m-carborane, in the β-1,3-glucan typified by schizophyllan, which is a natural polysaccharide, and arranging it in the internal space. The present invention has been derived.
Thus, the present invention provides a complex comprising β-1,3-glucan and carborane.
Furthermore, according to the present invention, there is provided a method for preparing the above-mentioned complex, which comprises a step of mixing β-1,3-glucan and carborane in a polar solvent solution and aging by adding water. Is done.

スエヒロタケと呼ばれるキノコから抽出されるシゾフィラン（以下SPGと記すことがある）は天然の状態では3重螺旋で存在するが、極性溶媒であるDMSOや、pH＞13以上のアルカリ水溶液では1本鎖のランダムコイルとして存在することが知られている（図３参照）。また、このランダムコイル状のSPGは溶媒を中性の水に戻すと再び３重螺旋の状態に巻き戻ることが知られている。この巻き戻りの過程に金ナノ粒子などの疎水性物質を共存させることにより、SPGはその螺旋内部にそれらの物質を取り込み、規則的に配列する。このSPGの特殊な性質を利用し、我々は現在までに様々な物質をその3重螺旋内部に取り込むことを明らかとした（非特許文献１４０、１５１）。
M. Numata, M.Asai, K. Kaneko, T. Hasegawa, N. Fujita, Y. Kitada, K. Sakurai, S. Shinkai,Chem. Lett., 232 (2004). 15 M. Numata,M. Asai, K. Kaneko, A. -H. Bae, T. Hasegawa, N. Fujita, K. Sakurai, S. Shinkai,J. Am. Chem. Soc., 127, 5875 (2005). Schizophyllan extracted from mushrooms called Suehirotake (hereinafter sometimes referred to as SPG) exists in a triple helix in the natural state, but it is a single-chain in DMSO, which is a polar solvent, or in an alkaline aqueous solution of pH> 13 or more. It is known to exist as a random coil (see FIG. 3). Further, it is known that this random coil-shaped SPG rewinds to a triple helical state again when the solvent is returned to neutral water. By making hydrophobic substances such as gold nanoparticles coexist in the rewinding process, SPG takes these substances inside the spiral and arranges them regularly. Using this special property of SPG, we have clarified that various substances have been incorporated into the triple helix (Non-Patent Documents 140 and 151).
M. Numata, M. Asai, K. Kaneko, T. Hasegawa, N. Fujita, Y. Kitada, K. Sakurai, S. Shinkai, Chem. Lett., 232 (2004). 15 M. Numata, M. Asai, K. Kaneko, A. -H. Bae, T. Hasegawa, N. Fujita, K. Sakurai, S. Shinkai, J. Am. Chem. Soc., 127, 5875 (2005 ).

SPGは天然の多糖であるため細胞親和性が高く、葉酸などの様々な細胞選択性部位を容易に導入することができる。このため、SPGとカルボランの複合化に成功すれば、細胞毒性が少なく、ガン細胞の周りに集積させるということも可能であると考えられる。なお、カルボランを包接する作用は、SPGと主鎖の構造が同じであるスクレログルカン、レンチナン、パッキマンおよびカードランのようなβ-1,3-グルカンに共通に認められる性質であり、これらも本発明において使用できるβ-1,3-グルカンである。   Since SPG is a natural polysaccharide, it has high cell affinity and can easily introduce various cell-selective sites such as folic acid. For this reason, if SPG and carborane are successfully combined, it is considered that there is little cytotoxicity and it can be accumulated around cancer cells. In addition, the action of clathrating carborane is a property commonly recognized in β-1,3-glucans such as scleroglucan, lentinan, packingman and curdlan, which have the same main chain structure as SPG. It is β-1,3-glucan that can be used in the present invention.

他方、カルボランとは、カルバボランとも呼ばれ、ポリボランの網目状の構造をもつボラン類の一部のホウ素原子が炭素原子で置き換えられたものの総称である。本発明において用いられるのに好適なカルボランは、図１に示されるように、正二十面体構造の、２つのホウ素原子が炭素原子に置き換わった構造から成る化学式B10C2H12で表され極めて安定なことで知られるm-カルボラン（m-Carborane）とo-カルボラン（o-Carborane）であり、特にm-カルボランが好ましい。m-カルボランとo-カルボランの構造の違いは、カルボラン中に存在する２つの炭素の位置関係にあり、m-カルボランはひとつの炭素ともうひとつの炭素の間にホウ素をひとつ挟んでいる形を呈し、o-カルボランはひとつの炭素のすぐ横にもうひとつの炭素が存在するような構造を有する。 On the other hand, carborane is also referred to as carbaborane, and is a general term for a part of boranes having a network structure of polyborane in which some boron atoms are replaced with carbon atoms. As shown in FIG. 1, a carborane suitable for use in the present invention is represented by the chemical formula B 10 C 2 H 12 having a structure of an icosahedral structure in which two boron atoms are replaced by carbon atoms. M-Carborane and o-Carborane, which are known to be extremely stable, and m-carborane is particularly preferred. The difference in structure between m-carborane and o-carborane lies in the positional relationship between the two carbons present in the carborane, and m-carborane has a form in which one boron is sandwiched between one carbon and another carbon. Presented, o-carborane has a structure in which another carbon exists right next to one carbon.

β-1,3-グルカンとカルボランから成る本発明の複合体は、β-1,3-グルカンとカルボランを極性溶媒中で混合し（β-1,3-グルカンおよびカルボランの各極性溶媒を混合したり、あるいは、極性溶媒中にβ-1,3-グルカンおよびカルボランのいずれか一方を溶解させた後、他方を添加混合するなど、いずれの手法でもよい）、水を加えて熟成するという簡便な方法で調製することができる。極性溶媒（非プロトン性極性溶媒）として特に好適なものはDMSO（ジメチルスルホキシド）であるが、DMFなど他の極性溶媒も使用可能である。熟成は、一般に、水を加えた溶液を室温下に1日〜3日間静置することにより行われる。
以下、本発明に従う複合体化の詳細な条件の例を実施例に沿って記述する。 The complex of the present invention consisting of β-1,3-glucan and carborane is prepared by mixing β-1,3-glucan and carborane in a polar solvent (mixing each polar solvent of β-1,3-glucan and carborane). Or by dissolving one of β-1,3-glucan and carborane in a polar solvent and then adding and mixing the other), and ripening by adding water Can be prepared by various methods. A particularly suitable polar solvent (aprotic polar solvent) is DMSO (dimethyl sulfoxide), but other polar solvents such as DMF can also be used. Aging is generally performed by allowing a solution to which water has been added to stand at room temperature for 1 to 3 days.
Hereinafter, examples of detailed conditions for complexation according to the present invention will be described with reference to Examples.

〔実施例１および比較例１〕
カルボランとシゾフィラン複合体溶液の調製 室温下条件下でm-カルボラン/DMSO溶液( 2 mg/mL)100μLとSPG/DMSO溶液（10mg/mL）100μLを混合し、水1800μLを加えることによってSPGを巻き戻した（溶液(丸１)）。この溶液を2日間室温条件下で静置した。溶液の最終濃度は[SPG]=7.76×10-4 mmol（モノマー単位）、[m-カルボラン]= 6.85×10-4mol/L、Vw（水/全溶媒容積比）=90%である。またm-カルボランの代わりにo-カルボランを用いて同様に溶液を調製した(溶液(丸２))。SPGの代わりに他の多糖（デキストラン、プルラン）を用いた溶液、多糖を含まないカルボランのみの溶液、SPGの巻き戻り過程が起こらない条件で調製したt-SPG溶液も比較例として調製した。溶液調製後、溶液(丸１)には沈殿が見られなかったが、他の溶液からは白い沈殿が確認された。 [Example 1 and Comparative Example 1]
Preparation of carborane and schizophyllan complex solution Mix 100µL of m-carborane / DMSO solution (2mg / mL) and 100µL of SPG / DMSO solution (10mg / mL) under room temperature condition, and roll SPG by adding 1800µL of water Returned (solution (circle 1)). This solution was allowed to stand at room temperature for 2 days. The final concentration of the solution is [SPG] = 7.76 × 10 −4 mmol (monomer unit), [m-carborane] = 6.85 × 10 −4 mol / L, Vw (water / total solvent volume ratio) = 90%. A solution was similarly prepared using o-carborane instead of m-carborane (solution (circle 2)). A solution using other polysaccharides (dextran, pullulan) instead of SPG, a solution containing only carborane without polysaccharides, and a t-SPG solution prepared under conditions that do not cause the SPG unwinding process were also prepared as comparative examples. After the solution preparation, no precipitate was observed in the solution (circle 1), but a white precipitate was confirmed from the other solutions.

〔実施例２および比較例〕
ICP-Msスペクトル測定（誘導プラズマ結合-マススペクトル測定）によるシゾフィランと複合化したm-カルボランの量の検討 メタノールによる再沈殿操作を行い、複合化していないm-カルボランを取り除いた。溶液を凍結乾燥させ、白色の固体を得た。これを超純水に溶解させ、ICP-Msスペクトル測定を行った。測定の結果、SPG／m-カルボラン複合体中にホウ素は12.4 wt%含まれているということがわかった。同時に、リファレンスとしてo-カルボランを用いたサンプルのICP-Msスペクトル測定を行ったところ、サンプルからホウ素の存在がほとんど確認されなかった（存在量0.04
wt%）。つまり、m-カルボランはSPGと複合化することによって水溶性が向上し、o-カルボランではSPGと相互作用しにくいことが判明した。 Example 2 and Comparative Example
Examination of amount of m-carborane complexed with schizophyllan by ICP-Ms spectrum measurement (inductive plasma coupling-mass spectrum measurement) Reprecipitation operation with methanol was performed to remove uncomplexed m-carborane. The solution was lyophilized to give a white solid. This was dissolved in ultrapure water, and ICP-Ms spectrum measurement was performed. As a result of the measurement, it was found that 12.4 wt% boron was contained in the SPG / m-carborane complex. At the same time, when ICP-Ms spectrum measurement of a sample using o-carborane as a reference was performed, the presence of boron was hardly confirmed in the sample (abundance 0.04).
wt%). In other words, it was found that m-carborane was improved in water solubility by complexing with SPG, and o-carborane was less likely to interact with SPG.

〔実施例３および比較例３〕
SPG存在溶液のIRスペクトル測定 SPGと複合化することによってm-カルボランのB-H 振動ピーク( 2596 cm-1)に違いがみられると考え、IRスペクトル測定を行った。測定結果を、m-カルボランから得られるIRスペクトル測定結果と比較することにより違いが見られることを期待した。SPG存在下で調製を行った溶液（丸１）に対し、メタノールによる再沈殿を行い、沈殿物を回収した。この操作により複合化していないm-カルボラン、SPGを除去した。その後、透析を行い、溶媒を完全に水に置換した。この溶液を凍結乾燥し、IRスペクトルを測定した。また、リファレンスとしてm-カルボランのみのIRスペクトルを測定し、IRスペクトルの比較を行った。図４のスペクトル測定の結果、SPGを含まないサンプルから得られたIRスペクトルにはB-Hの振動ピークが確認されなかったのに対し、SPG／m-カルボラン複合体のIRスペクトルはSPGとm-カルボランの足し合わせのIRスペクトルが確認された。またm-カルボランのみに比べ、B-Hの振動ピークが9cm-1程高エネルギーシフトしていることが確認された。複数回同じ操作で溶液調製、測定を行っても同様の結果が得られたことからも、この結果はSPGとm-カルボランが相互作用することにより分子振動に変化が生じたことを示している。また、この結果は過去の研究例とも一致している（非特許文献１２、１３)。m-カルボランの代わりにo-カルボランを用いたサンプルからはカルボラン由来のB-Hの振動ピークは確認されなかった。この結果は、SPGと相互作用する際にm-カルボランとo-カルボランで何らかの違いが起こっていることを示している。さらに他の多糖をSPGの代わりに用いた溶液も同様の操作を行い、B-Hの振動ピークの確認を行った。しかし、ほぼ全てのサンプルからはB-Hの振動ピークと思われるIRスペクトルは確認されなかった。このことからm-カルボランは他の多糖とは相互作用せずにSPGとのみ特異的に相互作用していることを示唆している。さらに巻き戻り過程を経ずにSPG とカルボランを混合したt-SPG溶液のサンプルからはB-Hの振動ピークは確認されなかった。このことからSPGとカルボランの相互作用はSPGの3重螺旋への巻き戻り過程が必要であることを示している。よって、カルボランはSPG内に取り込まれ相互作用することが判明した。
Frixa, M.Scobie, S. J. Black, A. S. Thompson, M. D. Threadgill, Chem. Commun., 2876(2002). P. D. Godfrey,W. J. Grigsby, P. J. Nichols. C. L. Raston, J. Am. Chem. Soc., 119, 9283(1997). Example 3 and Comparative Example 3
IR spectrum measurement of SPG presence solution It was thought that there was a difference in BH vibration peak (2596 cm-1) of m-carborane by combining with SPG, and IR spectrum measurement was performed. We expected to see the difference by comparing the measurement results with the IR spectrum measurement results obtained from m-carborane. The solution (circle 1) prepared in the presence of SPG was reprecipitated with methanol, and the precipitate was collected. By this operation, uncomplexed m-carborane and SPG were removed. Thereafter, dialysis was performed, and the solvent was completely replaced with water. This solution was freeze-dried and the IR spectrum was measured. In addition, IR spectra of only m-carborane were measured as a reference, and IR spectra were compared. As a result of spectrum measurement in FIG. 4, no BH vibration peak was confirmed in the IR spectrum obtained from the sample not containing SPG, whereas the IR spectrum of the SPG / m-carborane complex was SPG and m-carborane. As a result, an IR spectrum was confirmed. In addition, it was confirmed that the vibration peak of BH was shifted by about 9 cm -1 as compared with m-carborane alone. The same results were obtained even if the solution was prepared and measured multiple times with the same operation, and this result showed that the molecular vibration was changed by the interaction of SPG and m-carborane. . This result is consistent with past research examples (Non-patent Documents 12 and 13). From the sample using o-carborane instead of m-carborane, the vibration peak of BH derived from carborane was not confirmed. This result indicates that there is some difference between m-carborane and o-carborane when interacting with SPG. Furthermore, the same operation was performed on solutions using other polysaccharides in place of SPG, and the vibration peak of BH was confirmed. However, almost all samples did not show IR spectra that seem to be vibration peaks of BH. This suggests that m-carborane does not interact with other polysaccharides but specifically interacts only with SPG. Furthermore, no vibration peak of BH was confirmed from the sample of t-SPG solution in which SPG and carborane were mixed without going through the rewinding process. This suggests that the interaction between SPG and carborane requires a process of rewinding SPG to the triple helix. Thus, it was found that carborane is incorporated into SPG and interacts.
Frixa, M. Scobie, SJ Black, AS Thompson, MD Threadgill, Chem. Commun., 2876 (2002). PD Godfrey, WJ Grigsby, PJ Nichols. CL Raston, J. Am. Chem. Soc., 119, 9283 (1997).

〔実施例４および比較例４〕
TEM観察 m-カルボランとSPGの複合化によるモルフォロジー変化を見るため溶液を透析により溶媒を完全に水に置換した後、TEMグリッド（カーボン支持膜あり）にキャストし、TEM観察を行った。また、同時にそれぞれリファレンス溶液のTEM観察も行った。その結果、図５に示したようにSPGとの複合化操作を行った溶液からはファイバー状のモルフォロジーが確認されたが、SPGの存在しない溶液や、他の多糖を用いた溶液からはそのようなファイバー状の像が確認されなかった。また、m-カルボランの代わりにo-カルボランを用いた溶液からもファイバー状のモルフォロジーは確認されなかった。これらの結果からも、SPGはm -カルボランと特異的に相互作用していることが示された。 Example 4 and Comparative Example 4
TEM observation To observe the morphological change due to the combination of m-carborane and SPG, the solvent was completely replaced with water by dialysis, and then cast on a TEM grid (with carbon support film), and TEM observation was performed. At the same time, TEM observation of each reference solution was also performed. As a result, as shown in FIG. 5, a fiber-like morphology was confirmed from the solution subjected to the complexing operation with SPG, but from the solution containing no SPG or the solution using other polysaccharides. A fiber-like image was not confirmed. In addition, fiber morphology was not confirmed from a solution using o-carborane instead of m-carborane. These results also indicated that SPG interacted specifically with m-carborane.

〔実施例５および比較例５〕
AFM観察 m-カルボランとSPGが複合化することによるモルフォロジーの変化を確認するため、AFM観察を行った。SPG存在溶液とSPG非存在溶液から得られる結果を比較することで、SPGの存在によるモルフォロジーの変化が見られることを期待した。SPG存在溶液、およびSPG非存在溶液に対し透析を行った。この操作により溶媒を完全に水に置換した後、マイカ基盤にキャストし、減圧乾燥させた。各測定サンプルに対し、AFM観察を行った。AFM観察の結果、図６のようにSPG以外の多糖（プルランや、デキストラン、アミロース）溶液からは高さが不均一で4.5nm以上の凝集したような像が見られた。また、SPGを含むサンプルからは、高さの均一な像が確認された。AFMやTEM観察に像に見られるモルフォロジーから、SPGによってカルボランは均一な高さのファイバー状に配列制御されていることが示唆された。また、SPGを含むサンプルから得られたファイバー状モルフォロジーの高さは、2nm程度であることが確認された。SPG3重螺旋状態での高さはおよそ1nmであり、m-カルボラン１つの直径がおよそ1nmであることからすると、もし複合化しているならばSPG内でm-カルボランは1 次元的に配列されていることになる。ICP-Msスペクトル測定の結果から計算を行うと、m-カルボランは複合体中で糖と0.7(mol/mol)の比で複合化していることが認められ、複合体のICP-Msペクトル測定の結果とAFMから得られた平均的な高さはほぼ一致する。また、IRスペクトル測定によりSPGとm-カルボランの相互作用を示唆する結果が得られたことからもSPG内でm-カルボランは1次元的に配列していると考えられる（図７および図８）。 Example 5 and Comparative Example 5
AFM observation AFM observation was performed to confirm the change in morphology due to the combination of m-carborane and SPG. By comparing the results obtained from the SPG presence solution and the SPG non-existence solution, we expected to see a change in morphology due to the presence of SPG. Dialysis was performed on the SPG presence solution and the SPG absence solution. By this operation, the solvent was completely replaced with water, and then cast on a mica substrate and dried under reduced pressure. AFM observation was performed on each measurement sample. As a result of AFM observation, as shown in FIG. 6, a non-SPG polysaccharide (pullulan, dextran, amylose) solution with a nonuniform height and an aggregated image of 4.5 nm or more was observed. Further, an image having a uniform height was confirmed from the sample containing SPG. From the morphology observed in the images of AFM and TEM observations, it was suggested that carborane was arrayed and controlled by SPG in the form of a uniform fiber. The height of the fiber morphology obtained from the sample containing SPG was confirmed to be about 2 nm. The height in the SPG triple helix state is about 1 nm, and the diameter of one m-carborane is about 1 nm. If it is complex, m-carborane is arranged one-dimensionally in SPG. Will be. When calculated from the results of ICP-Ms spectrum measurement, m-carborane was found to be complexed with the sugar at a ratio of 0.7 (mol / mol) in the complex, and the ICP-Ms spectrum measurement of the complex was confirmed. The average height obtained from AFM agrees with the result. Moreover, since the results suggesting the interaction between SPG and m-carborane were obtained by IR spectrum measurement, m-carborane is considered to be one-dimensionally arranged in SPG (FIGS. 7 and 8). .

カルボランを天然の多糖であるSPG等のβ-1,3-グルカンで被覆することによってカルボランの水溶性を向上させることを見出した。β-1,3-グルカンが選択的にカルボランを取り込むことによってβ-1,3-グルカンとカルボランから成る複合体が形成され、この複合体を利用することにより、生体適応性の増加などガン細胞へのカルボランのデリバリーシステムの構築が期待される。   It was found that the water solubility of carborane was improved by coating carborane with β-1,3-glucan such as SPG which is a natural polysaccharide. β-1,3-glucan selectively takes up carborane to form a complex consisting of β-1,3-glucan and carborane. By using this complex, cancer cells such as increased biocompatibility It is expected that a carborane delivery system will be constructed.

カルボランの構造（左：o-体、右：m-体）を示す。Shows the structure of carborane (left: o-isomer, right: m-isomer). カルボランの熱中性子線吸収とα線放射を模式的に示す。The thermal neutron absorption and α-ray emission of carborane are schematically shown. SPGの3重螺旋とランダムコイルを模式的に示す。An SPG triple helix and a random coil are schematically shown. SPG／カルボラン系のIR スペクトルを示す： (A) SPG／m-カルボラン、(B) SPG／o-カルボラン、 (C) 3重鎖SPG／m-カルボラン（実施例３および比較例３）。The IR spectra of the SPG / carborane system are shown: (A) SPG / m-carborane, (B) SPG / o-carborane, (C) triple-chain SPG / m-carborane (Example 3 and Comparative Example 3). TEM観察結果を示す：(A) SPG存在溶液、(B) SPG非存在溶液（実施例４および比較例４）The TEM observation results are shown: (A) SPG presence solution, (B) SPG absence solution (Example 4 and Comparative Example 4) AFM観察結果を示す：(A) SPG存在溶液、(B) SPG非存在溶液（実施例５および比較例５）The AFM observation results are shown: (A) SPG presence solution, (B) SPG absence solution (Example 5 and Comparative Example 5) SPGとカルボランの相互作用模式図（実施例５および比較例５）を示す。The interaction schematic diagram (Example 5 and Comparative Example 5) of SPG and carborane is shown. SPG／カルボラン複合体形成模式図（実施例５および比較例５）を示す。The SPG / carborane complex formation schematic diagram (Example 5 and Comparative Example 5) is shown.

Claims (4)

β-1,3-グルカンとカルボランから成ることを特徴とする複合体。   A complex comprising β-1,3-glucan and carborane. カルボランがm-カルボランであることを特徴とする請求項１の複合体。   The complex of claim 1, wherein the carborane is m-carborane. β-1,3-グルカンがシゾフィラン、スクレログルカン、レンチナン、パッキマンまたはカードランから選ばれたものであることを特徴とする請求項１の複合体。   The complex according to claim 1, wherein the β-1,3-glucan is selected from schizophyllan, scleroglucan, lentinan, Pacquiman or curdlan. β-1,3-グルカンおよびカルボランを極性溶媒溶液中で混合し、水を加えて熟成する工程を含むことを特徴とすることにより、請求項１の複合体を調製する方法。
A method for preparing the complex according to claim 1, comprising the steps of mixing β-1,3-glucan and carborane in a polar solvent solution and aging by adding water.
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