JP6281991B2 - Carbon nanohorn carrying boron compound on inner and outer walls and method for producing the same - Google Patents
Carbon nanohorn carrying boron compound on inner and outer walls and method for producing the same Download PDFInfo
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- JP6281991B2 JP6281991B2 JP2016512786A JP2016512786A JP6281991B2 JP 6281991 B2 JP6281991 B2 JP 6281991B2 JP 2016512786 A JP2016512786 A JP 2016512786A JP 2016512786 A JP2016512786 A JP 2016512786A JP 6281991 B2 JP6281991 B2 JP 6281991B2
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Description
本発明は、ホウ素化合物を内包および外壁に担持するカーボンナノホーン及びその製造方法に関し、特に、ホウ素中性子捕捉療法(ＢＮＣＴ：Boron Neutron Capture Therapy)に用いるホウ素化合物を内包および外壁に担持するカーボンナノホーン及びその製造方法に関するものである。 TECHNICAL FIELD The present invention relates to a carbon nanohorn that supports a boron compound on an inner wall and an outer wall, and a method for producing the same. It relates to a manufacturing method.
ホウ素中性子捕捉療法は、正常組織よりもがん組織に集合しやすいホウ素化合物を、あらかじめ癌細胞内に取り込ませておき、外部からエネルギーの低い熱中性子線を照射すると、ホウ素の原子核が低速の熱中性子を好んで捕捉し、捕捉と同時に核***を起こし、α粒子を発生し、このα粒子で近傍のがん細胞を殺傷させる手法である。 In boron neutron capture therapy, boron compounds that are more likely to collect in cancer tissues than normal tissues are taken into cancer cells in advance and irradiated with thermal neutrons with low energy from the outside. This is a technique that favors neutron capture, causes fission at the same time as capture, generates alpha particles, and kills nearby cancer cells with the alpha particles.
ホウ素中性子捕捉療法では、腫瘍に対して高い親和性と選択性を持ち、かつ正常組織に対する蓄積・損傷を最小限にすることが可能なホウ素製剤が必須であるが、現在使われている、ホウ素フェニルアラニンやオルトデカボランなどでは、化学的安定性が十分でないことや腫瘍への蓄積量を上げることが困難であるなどの問題があり、ホウ素中性子捕捉療法の効果を高めることができない。 Boron neutron capture therapy requires a boron preparation that has high affinity and selectivity for tumors and that can minimize accumulation and damage to normal tissues. Phenylalanine, orthodecaborane, and the like have problems such as insufficient chemical stability and difficulty in increasing the amount accumulated in the tumor, and the effect of boron neutron capture therapy cannot be enhanced.
近年、ホウ素中性子捕捉療法用製剤の性能向上を目指して多くの研究が進められるようになり、ポルフィリンやフラーレン(特許文献１参照)、あるいはヘテロポリ酸(特許文献２参照)などの化合物中にホウ素を導入した例、リポソーム中にＢＳＨやＢＰＡを導入したもの(特許文献３および４参照)、カルボランのような無機系ホウ素化合物(特許文献５参照)の利用、ポリマーにＢＰＡ等を含む化合物を反応させたもの(特許文献６参照)などの複合系を利用しようとする試みも行われている。しかし、これらのいずれの手法も、ホウ素の量を飛躍的に向上することは不可能である。
In recent years, a lot of research has been promoted with the aim of improving the performance of preparations for boron neutron capture therapy, and boron is contained in compounds such as porphyrin, fullerene (see Patent Document 1), or heteropolyacid (see Patent Document 2). Examples of introduction, those in which BSH or BPA is introduced into liposomes (see
一方、ＢＮナノチューブを内包したカーボンナノチューブが報告されている(非特許文献１参照)。しかしながら、該非特許文献１には、ホウ素中性子捕捉療法用製剤としての利用については、触れられていない。
また、カーボンナノチューブでは、直径が細いために内包量を上げることが困難である。また、カーボンナノチューブは、チューブ同士がファンデルワールス力で凝集し、分散させることが困難であるという欠点を有する。On the other hand, carbon nanotubes containing BN nanotubes have been reported (see Non-Patent Document 1). However, the
Further, since the diameter of the carbon nanotube is small, it is difficult to increase the amount of inclusion. Carbon nanotubes also have the disadvantage that the tubes are agglomerated by van der Waals forces and difficult to disperse.
前述のとおり、これまでの、ホウ素中性子捕捉療法に使われているホウ素製剤は、腫瘍蓄積量が少ないという欠点を有する。
本発明は、こうした現状を鑑みてなされたものであって、ホウ素の腫瘍蓄積量が多いホウ素製剤を提供することを目的とするものである。As described above, conventional boron preparations used for boron neutron capture therapy have the disadvantage that the tumor accumulation is small.
This invention is made | formed in view of such the present condition, Comprising: It aims at providing the boron formulation with many tumor accumulation amounts of boron.
本発明者らは、上記目的を達成すべく鋭意研究を重ねた結果、中性子捕捉療法用製剤として、ホウ素化合物を内包および外壁に担持するカーボンナノホーンを用いることにより、ホウ素化合物を腫瘍細胞へ選択的に送達し、ホウ素の腫瘍蓄積量を上げることが可能になるという知見を得た。 As a result of intensive studies to achieve the above-mentioned object, the present inventors have selected boron compounds as tumor cells by using carbon nanohorns carrying boron compounds on the inner and outer walls as neutron capture therapy preparations. We have obtained the knowledge that it is possible to increase the tumor accumulation amount of boron.
本発明はこれらの知見に基づいて完成に至ったものであり、本発明によれば、以下の発明が提供される。
［１］ホウ素化合物が内包及びおよび外壁に担持されていることを特徴とするカーボンナノホーン。
［２］前記ホウ素化合物が、窒化ホウ素であることを特徴とする[１]に記載のカーボンナノホーン。
［３］ホウ素化合物の原料にアンモニアボランを用い、カーボンナノホーンとともに加熱することを特徴とする、ホウ素化合物が内包および外壁に担持されているカーボンナノホーンの製造方法。
［４］７５０℃以上に加熱することを特徴とする[３]に記載の、ホウ素化合物が内包および外壁に担持されているカーボンナノホーンの製造方法。
［５］[１]又は[２]に記載のカーボンナノホーンの表面に、抗体または葉酸を付加させたリン脂質ＰＥＧが物理的に付着して表面修飾されていることを特徴とするカーボンナノホーン複合体。
［６］[１]又は[２]に記載のカーボンナノホーンに導入された官能基に、抗体付加ポリエチレングリコールが化学結合されていることを特徴とするカーボンナノホーン複合体。
［７］[１]又は[２]に記載のカーボンナノホーン或いは[５]又は[６]に記載のカーボンナノホーン複合体を有効成分として含有することを特徴とするホウ素中性子捕捉療法用製剤。The present invention has been completed based on these findings, and according to the present invention, the following inventions are provided.
[1] A carbon nanohorn characterized in that a boron compound is supported on the inner and outer walls.
[2] The carbon nanohorn according to [1], wherein the boron compound is boron nitride.
[3] A method for producing a carbon nanohorn having a boron compound supported on an inner wall and an outer wall, wherein ammonia borane is used as a raw material for the boron compound and heated together with the carbon nanohorn.
[4] The method for producing carbon nanohorns according to [3], wherein the boron compound is supported on the inner wall and the outer wall, which is heated to 750 ° C. or higher.
[5] A carbon nanohorn complex, wherein the surface of the carbon nanohorn according to [1] or [2] is modified by physically attaching a phospholipid PEG to which an antibody or folic acid is added. .
[6] A carbon nanohorn complex characterized in that antibody-added polyethylene glycol is chemically bonded to the functional group introduced into the carbon nanohorn according to [1] or [2].
[7] A boron neutron capture therapy preparation comprising the carbon nanohorn according to [1] or [2] or the carbon nanohorn complex according to [5] or [6] as an active ingredient.
本発明のホウ素化合物を内包および外壁に担持しているカーボンナノホーン(以下、「ＢＮ−ＣＮＨ」ということもある。)は、カーボンナノホーン(以下、「ＣＮＨ」ということもある。)の直径が２〜５ｎｍと大きいために、大量のホウ素化合物を内包しており、ホウ素の腫瘍蓄積量を上げることが可能になる。また、比表面積が３００−４００ｍ２／ｇであるため、外壁にも大量のホウ素化合物を担持できる。特に、原料としてアンモニアボラン(Ｈ３Ｎ−ＢＨ３)を用いた場合、加熱によりアンモニアボランからＢＮへ変化し、その際にＢＮがＣＮＨ内部や外壁にとどまることで、BN−CNHをえることができる。
また、ＣＮＨは化学的に安定であって、その外壁を修飾することが容易であるので、本発明のＢＮ−ＣＮＨを化学的にあるいは物理的に修飾することにより、血中投与した際の腫瘍での蓄積量を上げることが可能となる。
さらに、本発明に用いるＣＮＨは、１００ｎｍ程度の球状構造をとっていて、しかも表面は凹凸に富んだ構造をしているため、強く凝集することはなく、溶液中にて孤立分散する。したがって、適切に表面修飾したＢＮ−ＣＮＨは、生体内投与した際に、腫瘍への蓄積を実現させ、ホウ素中性子捕捉療法の効果を高めることができる。The carbon nanohorn (hereinafter also referred to as “BN-CNH”) carrying the boron compound of the present invention on the inner wall and the outer wall has a carbon nanohorn (hereinafter also referred to as “CNH”) diameter of 2. Since it is as large as ˜5 nm, a large amount of boron compound is included, and the tumor accumulation amount of boron can be increased. Moreover, since a specific surface area is 300-400 m < 2 > / g, a large amount of boron compounds can be carry | supported also on an outer wall. In particular, when ammonia borane (H 3 N—BH 3 ) is used as a raw material, BN-CNH can be obtained by changing from ammonia borane to BN by heating, and at that time BN stays inside or outside the CNH. it can.
Moreover, since CNH is chemically stable and it is easy to modify its outer wall, the tumor when administered in blood by chemically or physically modifying the BN-CNH of the present invention It is possible to increase the amount of storage in
Furthermore, CNH used in the present invention has a spherical structure of about 100 nm, and the surface is rich in unevenness, so that it does not agglomerate strongly and is isolated and dispersed in the solution. Accordingly, appropriately surface-modified BN-CNH can realize accumulation in a tumor and enhance the effect of boron neutron capture therapy when administered in vivo.
本発明は、カーボンナノホーンにホウ素化合物が内包および外壁に担持されていることを特徴とするものである。 The present invention is characterized in that a boron compound is supported on an inner wall and an outer wall of a carbon nanohorn.
本発明におけるカーボンナノホーンの製造方法は、特に限定されず、ＣＶＤ法、レーザーアブレーション法、アーク放電法など、いずれの方法であっても構わない。 The method for producing the carbon nanohorn in the present invention is not particularly limited, and any method such as a CVD method, a laser ablation method, or an arc discharge method may be used.
本発明において、カーボンナノホーンに内包および外壁に担持させるホウ素化合物及びその方法は、特に限定されないが、たとえば、原料にアンモニアボランを用い、ＣＮＨとともに加熱する方法があげられる。該方法によれば、加熱によりアンモニアボランからＢＮへ変化し、その際にＢＮがＣＮＨ内部や外壁にとどまることで、BN−CNHを得ることができる。
ちなみに、化学反応式は、以下のとおりである。
ＮＨ3ＢＨ3 → ＢＮ＋３Ｈ2 In the present invention, the boron compound to be supported on the inner wall and the outer wall of the carbon nanohorn and the method thereof are not particularly limited, and examples thereof include a method in which ammonia borane is used as a raw material and heated together with CNH. According to this method, BN-CNH can be obtained by changing from ammonia borane to BN by heating, and at that time BN stays inside or outside the CNH.
Incidentally, the chemical reaction formula is as follows.
NH 3 BH 3 → BN + 3H 2
カーボンナノホーンは化学的に安定であって、その外壁を化学修飾することが容易であるので、血中投与した際に腫瘍での蓄積量を上げるために、本発明のＢＮ−ＣＮＨを化学的にあるいは物理的に修飾することができる。 Since carbon nanohorn is chemically stable and it is easy to chemically modify its outer wall, the BN-CNH of the present invention is chemically added to increase the amount of accumulation in the tumor when administered in blood. Alternatively, it can be physically modified.
化学的な修飾の一例として、例えば、本発明のＢＮ−ＣＮＨに、ＰＥＧを介して、がん細胞にその受容体が多数形成されている上皮成長因子(ＥＧＦ：Epidermal Growth Factor)を結合させることで、ＢＮ−ＣＮＨをがん細胞に特異的に集積させることができる。
具体的には、本発明のＢＮ−ＣＮＨを酸化処理してカルボキシル基を導入した後、得られたＢＮ−ＣＮＨのカルボキシル基にアミド結合によりＰＥＧ−ＣＯＯＨを付加して、ＢＮ−ＣＮＨ-ＰＥＧ−ＣＯＯＨを得、得られたＢＮ−ＣＮＨ−ＰＥＧ−ＣＯＯＨに、Ｎ−ヒドロキシコハク酸イミド(ＮＨＳ)とＥＤＣを少量加えて、ＢＮ−ＣＮＨ−ＰＥＧ−ＮＨＳを得た後、トリエチルアミンとＥＧＦを加えて、ＢＮ−ＣＮＨ−ＰＥＧ−ＥＧＦを得ることができる。As an example of chemical modification, for example, epidermal growth factor (EGF) in which a large number of receptors are formed in cancer cells is bound to BN-CNH of the present invention via PEG. Thus, BN-CNH can be accumulated specifically in cancer cells.
Specifically, the BN-CNH of the present invention was oxidized to introduce a carboxyl group, PEG-COOH was added to the carboxyl group of the obtained BN-CNH by an amide bond, and BN-CNH-PEG- COOH was obtained, and a small amount of N-hydroxysuccinimide (NHS) and EDC was added to the obtained BN-CNH-PEG-COOH to obtain BN-CNH-PEG-NHS, and then triethylamine and EGF were added. BN-CNH-PEG-EGF can be obtained.
また、物理的な修飾の一例として、例えば、本発明のＢＮ−ＣＮＨの表面に、ＰＬＰＥＧ−ＦＡを物理的に結合させることで、ＢＮ−ＣＮＨをがん細胞に特異的に集積させることができる。
すなわち、がん細胞には葉酸受容体が多数形成されているため、リン脂質ＰＥＧ(ＰＬＰＥＧ)と葉酸(ＦＡ)を反応さて、得られたＰＬＰＥＧ−ＦＡをカーボンナノチューブにファンデルワールス力により物理的に付着させるとカーボンナノチューブががん細胞に集積しやすくなることが示されており(飯泉陽子、博士学理論文、筑波大学、２０１２年参照)、同様な手法により、ＰＬＰＥＧ−ＦＡを、本発明のＢＮ−ＣＮＨの表面に物理的に結合させると、ＢＮ−ＣＮＨをがん細胞に特異的に集積させることが可能となる。In addition, as an example of physical modification, for example, BN-CNH can be specifically accumulated in cancer cells by physically binding PLPEG-FA to the surface of BN-CNH of the present invention. .
That is, since many folate receptors are formed in cancer cells, phospholipid PEG (PLPEG) and folic acid (FA) are reacted, and the resulting PLPEG-FA is physically converted into carbon nanotubes by van der Waals force. It has been shown that carbon nanotubes are likely to accumulate in cancer cells when attached to (see Yoko Iizumi, Ph.D. theory, University of Tsukuba, 2012). When physically bound to the surface of BN-CNH, BN-CNH can be specifically accumulated in cancer cells.
以下に、リン脂質ＰＥＧ(ポリ(エチレングリコール)誘導体化ジステアロイルホスファチジルエタノールアミン、以下、「ＰＬＰＥＧ」とする。)、葉酸(ＦＡ)、及びその化合物(ＰＬＰＥＧ−ＦＡ)の分子構造と反応式を示す(前掲論文、図５−９参照)。 The molecular structures and reaction formulas of phospholipid PEG (poly (ethylene glycol) derivatized distearoylphosphatidylethanolamine, hereinafter referred to as “PLPEG”), folic acid (FA), and its compound (PLPEG-FA) are shown below. This is shown (see the above paper, Fig. 5-9).
以下、本発明を実施例に基づいて説明するが、本発明はこの実施例に限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated based on an Example, this invention is not limited to this Example.
（ＢＮ−ＣＮＨの作製）
ＣＮＨは乾燥空気中で室温から５００℃まで１℃／ｍｉｎで昇温し、その後放冷して開孔させた。アンモニアボラン１ｍｇと開孔ＣＮＨ１ｍｇをガラス容器にいれて、４×１０-4Ｐａにて２時間真空排気後、ガラス容器の一端を封じ切ってアンプルを得た。
得られた試料入りアンプルを電気炉に入れ、２４時間かけて所定の温度(５５０℃から８５０℃までの、５０℃毎に異なる温度)まで昇温し、２４時間保った後、放冷させた。この処理により、ＢＮ−ＣＮＨを得た。
アンプルを開封して、ＢＮ−ＣＮＨを取り出し、エタノールで遊離している余剰なホウ素化合物をフィルターろ過することで洗い流した後、構造評価に用いた。(Production of BN-CNH)
CNH was heated at 1 ° C./min from room temperature to 500 ° C. in dry air, and then allowed to cool to open holes. Ammonia borane (1 mg) and open-hole CNH (1 mg) were placed in a glass container, evacuated at 4 × 10 −4 Pa for 2 hours, and then one end of the glass container was sealed to obtain an ampoule.
The obtained ampule with sample was put in an electric furnace, heated to a predetermined temperature (a temperature varying from 550 ° C. to 850 ° C. every 50 ° C.) over 24 hours, kept for 24 hours, and then allowed to cool. . By this treatment, BN-CNH was obtained.
The ampoule was opened, BN-CNH was taken out, and the excess boron compound liberated with ethanol was washed away by filtering, and then used for structural evaluation.
（誘導結合プラズマ(ＩＣＰ)発光測定）
各温度で合成したＢＮ−ＣＮＨを、それぞれ、１％ドデシルベンゼンスルホン酸ナトリウム水溶液中に０.０５ｍｇ／ｍＬになるように加え、チップ型ソニケーションで５分間処理し、ＢＮ−ＣＮＨ分散溶液を得た。これをＩＣＰ発光測定することにより、ＢＮ−ＣＮＨ中のホウ素含有量を定量した。その結果を図１ａに示す。
また、ＢＮ−ＣＮＨ(１ｍｇ)を、リン酸緩衝生理食塩水(ＰＢＳ)(２ｍＬ)及び細胞培養液(Cell Culture Meddium)(２ｍＬ)にそれぞれ加え、超音波洗浄機を用いて５分間分散させた後、３日間静置させた。３日間静置後、濾過によりＢＮ−ＣＮＨを回収し、ＢＮ−ＣＮＨに残っていたホウ素量を上述の方法でＩＣＰにて測定して見積もった。ＢＮ−ＣＮＨ中のホウ素量は、浸漬前のホウ素量に対する％で示すと、それぞれ図１ｂ、ｃのようになった。
図１ｂ、ｃに示すとおり、７５０℃以上で加熱処理したＢＮ−ＣＮＨが安定した構造をとると考えられたため、以後は、８００℃で加熱処理したＢＮ−ＣＮＨを構造評価に用いた。
８００℃で作製したＢＮ−ＣＮＨ(１ｍｇ)を細胞培養液(２ｍＬ)に加え、超音波洗浄機にて５分間分散させた後、０〜１０日間静置させた。静置後、濾過によりＢＮ−ＣＮＨを回収し、ＢＮ−ＣＮＨに残っていたホウ素量を上述の方法でＩＣＰにて測定して見積もった。ＢＮ−ＣＮＨ中のホウ素量は、浸漬前のホウ素量に対する％で示すと図１ｄのようになった。ＢＮ−ＣＮＨ中のホウ素量は、１０日間浸漬でも浸漬前のホウ素量の７５−８０％に保たれることが分かった。(Inductively coupled plasma (ICP) emission measurement)
Add BN-CNH synthesized at each temperature to 0.05 mg / mL in 1% sodium dodecylbenzenesulfonate aqueous solution and treat with chip-type sonication for 5 minutes to obtain a BN-CNH dispersion. It was. By measuring this with ICP luminescence, the boron content in BN-CNH was quantified. The result is shown in FIG.
BN-CNH (1 mg) was added to phosphate buffered saline (PBS) (2 mL) and cell culture medium (2 mL), respectively, and dispersed for 5 minutes using an ultrasonic cleaner. Thereafter, it was allowed to stand for 3 days. After standing for 3 days, BN-CNH was recovered by filtration, and the amount of boron remaining in BN-CNH was measured by ICP by the above method and estimated. When the boron content in BN-CNH is expressed as a percentage of the boron content before immersion, the results are as shown in FIGS. 1b and 1c, respectively.
As shown in FIGS. 1b and 1c, since it was considered that BN-CNH heat-treated at 750 ° C. or higher had a stable structure, BN-CNH heat-treated at 800 ° C. was used for structural evaluation.
BN-CNH (1 mg) prepared at 800 ° C. was added to the cell culture medium (2 mL), dispersed for 5 minutes with an ultrasonic washer, and allowed to stand for 0 to 10 days. After standing, BN-CNH was recovered by filtration, and the amount of boron remaining in BN-CNH was measured and estimated by ICP using the method described above. The amount of boron in BN-CNH was as shown in FIG. 1d as a percentage of the amount of boron before immersion. It was found that the boron content in BN-CNH was maintained at 75-80% of the boron content before immersion even after 10 days immersion.
（透過型電子顕微鏡観察(ＴＥＭ)と電子エネルギー損失分光(ＥＥＬＳ)測定）
得られた前記ＢＮ−ＣＮＨをエタノールに分散させ、ＴＥＭ観察とＥＥＬＳ測定を行った。
ＴＥＭ測定の結果を、図２ａ−ｄに示す。図２ａ−ｄに示すとおり、ＢＮ−ＣＮＨはＣＮＨ個々の擬円筒状構造とその球状集合体形状を維持していた。さらに高倍率で観察してみると、ＣＮＨの壁に沿って、壁の内側と外側に層状にＢＮが合成されていることがわかった。また、ＣＮＨ内部空間にもＢＮが存在していることを確認した。ＢＮは六方晶ＢＮとして存在していると推知される。図２ｅに示すＥＥＬＳスペクトルには、Ｂ、Ｎ、及びＣによるピークが観察され、Ｂ及びＮのピーク形状は六方晶ＢＮのものと一致した。(Transmission electron microscope observation (TEM) and electron energy loss spectroscopy (EELS) measurement)
The obtained BN-CNH was dispersed in ethanol, and TEM observation and EELS measurement were performed.
The results of TEM measurement are shown in FIGS. 2a-d. As shown in FIGS. 2a to 2d, BN-CNH maintained the pseudo-cylindrical structure of each CNH and its spherical aggregate shape. When observed at a higher magnification, it was found that BN was synthesized in layers on the inside and outside of the wall along the CNH wall. It was also confirmed that BN was present in the CNH internal space. It is presumed that BN exists as hexagonal BN. In the EELS spectrum shown in FIG. 2e, peaks due to B, N, and C were observed, and the peak shapes of B and N coincided with those of hexagonal BN.
（Ｘ線光電子分光(ＸＰＳ)測定）
前記のＢＮ−ＣＮＨ(１ｍｇ)とエタノール(５ｍＬ)を混合し、５分間バスソニケーションして得た分散液をＳｉウエハー上に滴下し、乾燥させた後に、ＸＰＳ測定を行った。結果を図３に示す。
図３に示すとおり、ＢＮ−ＣＮＨは、Ｂ、Ｃ、及びＮに帰属されるピークが観察され、それぞれの強度から、Ｂ(２２％)、Ｃ(５５％)、Ｎ(２２％)であることが分かった(％は原子数％)。酸素のピークも見られたが、これは、基盤に用いたＳｉウエハーの表面のＳｉ酸化物による。(X-ray photoelectron spectroscopy (XPS) measurement)
The dispersion obtained by mixing BN-CNH (1 mg) and ethanol (5 mL) and bath sonicating for 5 minutes was dropped on a Si wafer and dried, and then XPS measurement was performed. The results are shown in FIG.
As shown in FIG. 3, in BN-CNH, peaks attributed to B, C, and N are observed, and from the respective intensities, they are B (22%), C (55%), and N (22%). (% Is atomic%). An oxygen peak was also observed, but this was due to the Si oxide on the surface of the Si wafer used for the substrate.
（動的光散乱測定(ＤＬＳ)）
前記のＢＮ−ＣＮＨ(１ｍｇ)をＰＬＰＥＧのリン酸緩衝生理食塩水(ＰＢＳ)(５ｍＬ)に加え、バスソニケーションで、５分間ソニケーションしてＢＮ−ＣＮＨを分散させた後に、粒子径をＤＬＳにて測定した。比較のため、同様にして前記の開孔したＣＮＨ(１ｍｇ)を分散させた後に、粒子径をＤＬＳにて測定した。それぞれの結果を、図４のｂ、ａに示す。
ｂに示すＢＮ−ＣＮＨの平均粒子径は約１３０ｎｍであり、ａに示すもとのＣＮＨの平均粒子径約１２０ｎｍとほぼ一致し、ホウ素化合物を内包および外壁に担持する操作により構造が大きく変わらなかったことを示唆している。電子顕微鏡観察によりＣＮＨの平均直径が約１００ｎｍであることが知られているので、ＰＬＰＥＧでＰＢＳ中に分散したＢＮ−ＣＮＨはほぼ孤立に分散していることが分かる。また、この分散状態は少なくとも数日間は安定であった。(Dynamic light scattering measurement (DLS))
After adding BN-CNH (1 mg) to PLPEG phosphate buffered saline (PBS) (5 mL) and sonicating with bath sonication for 5 minutes to disperse BN-CNH, the particle size was changed to DLS. Measured. For comparison, the above-mentioned CNH (1 mg) having the pores was dispersed in the same manner, and then the particle size was measured by DLS. Each result is shown in FIG.
The average particle size of BN-CNH shown in b is about 130 nm, which is almost the same as the average particle size of about 120 nm of the original CNH shown in a, and the structure is not greatly changed by the operation of supporting the boron compound on the inner wall and the outer wall. It suggests that. Since the average diameter of CNH is known to be about 100 nm by electron microscope observation, it can be seen that BN-CNH dispersed in PBS with PLPEG is almost isolated. This dispersion state was stable for at least several days.
（ＢＮ−ＣＮＨの表面修飾）
腫瘍へＢＮ−ＣＮＨを高濃度蓄積させるために、ＢＮ−ＣＮＨ表面をポリエチレングリコール(ＰＥＧ)によりコーティングしてマクロファージによる貪食を避け、さらに、葉酸(ＦＡ)をＢＮ−ＣＮＨに付加して、ＢＮ−ＣＮＨをがん細胞選択的に取り込ませるようにする。
そのためにまず、ＦＡ(１ｍｇ)をＤＭＳＯ(４ｍＬ)に溶解させ、この溶液に、ＰＬＰＥＧ(２００ｍｇ)、ピリジン(２ｍＬ)、ＥＤＣ(３８ｍｇ)を加え、４時間室温にて撹拌した。ロータリーエバポレーターでピリジンを蒸発させた後に水５０ｍＬを加えた。得られた液を透析膜(ＭＷＣＯ３５００)に入れ、水２Ｌ中に浸したのちに室温にて放置した。約１日後に、水２Ｌを入れ替えるという操作を５回行い、未反応ＰＬＰＥＧ分子を除去した。得られた液を凍結乾燥させ、ＰＬＰＥＧ−ＦＡの粉末を得た。ＰＬＰＥＧ−ＦＡの構造は1Ｈ ＮＭＲにて確認した。(Surface modification of BN-CNH)
In order to accumulate high concentrations of BN-CNH in the tumor, the surface of BN-CNH is coated with polyethylene glycol (PEG) to avoid phagocytosis by macrophages, and folic acid (FA) is added to BN-CNH to add BN- CNH is selectively taken up by cancer cells.
For that purpose, FA (1 mg) was first dissolved in DMSO (4 mL), PLPEG (200 mg), pyridine (2 mL), EDC (38 mg) were added to this solution, and the mixture was stirred at room temperature for 4 hours. After evaporating pyridine on a rotary evaporator, 50 mL of water was added. The obtained liquid was put into a dialysis membrane (MWCO 3500), immersed in 2 L of water, and allowed to stand at room temperature. About 1 day later, 2 L of water was replaced five times to remove unreacted PLPEG molecules. The obtained liquid was freeze-dried to obtain a PLPEG-FA powder. The structure of PLPEG-FA was confirmed by 1 H NMR.
得られたＰＬＰＥＧ−ＦＡでＢＮ−ＣＮＨ表面を修飾するため、ＰＬＰＥＧ−ＦＡ(４ｍｇ)と、前記ＢＮ−ＣＮＨ(３ｍｇ)をリン酸緩衝生理食塩水(ＰＢＳ)(２ｍＬ)中にて混合させ、ＰＬＰＥＧ−ＦＡの脂質基(アルキル鎖)をＢＮ−ＣＮＨの表面にワンデルワール力で結合させ、がん細胞選択的に取り込まれるＢＮ−ＣＮＨ／ＰＬＰＥＧ−ＦＡを合成した。 In order to modify the BN-CNH surface with the obtained PLPEG-FA, PLPEG-FA (4 mg) and the BN-CNH (3 mg) were mixed in phosphate buffered saline (PBS) (2 mL), The lipid group (alkyl chain) of PLPEG-FA was bonded to the surface of BN-CNH with a one-delwar force to synthesize BN-CNH / PLPEG-FA that is selectively taken up by cancer cells.
ＣＮＨをＰＬＰＥＧ−ＦＡを用いてＰＢＳ中に分散させたもの(ＣＮＨ／ＰＬＰＥＧ−ＦＡ)と、［００２８］で得られたＢＮ−ＣＮＨ／ＰＬＰＥＧ−ＦＡのＰＢＳ分散液における粒子サイズをＤＬＳにて測定した。その結果をそれぞれ、図４のｄ、ｃに示す。
ｄに示すＢＮ−ＣＮＨ／ＰＬＰＥＧ−ＦＡの平均粒子サイズは約１３０ｎｍであり、上記［００２６］と同様、ｃに示すもとのＣＮＨのサイズとほぼ一致し、ＦＡ付加後においても、表面修飾したＣＮＨは単一分散していることがわかる。Particle size in PBS dispersion of CNH dispersed in PBS using PLPEG-FA (CNH / PLPEG-FA) and BN-CNH / PLPEG-FA obtained in [0028] was measured with DLS. did. The results are shown in FIG. 4 d and c, respectively.
The average particle size of BN-CNH / PLPEG-FA shown in d is about 130 nm, and almost the same as the size of the original CNH shown in c, as in the above [0026], and surface modification was performed even after the addition of FA. It can be seen that CNH is monodispersed.
（がん細胞のＢＮ−ＣＮＨ／ＰＬＰＥＧ−ＦＡ選択的取り込み実験）
ヒト口腔類上皮がん由来の細胞株(ＫＢ細胞)を培養し、そこに、ＰＢＳに分散させたＢＮ−ＣＮＨ／ＰＬＰＥＧ−ＦＡ溶液を、最終濃度１０μｇ／ｍＬになるように添加した。２４時間培養後、細胞に取り込まれなかったＢＮ−ＣＮＨ／ＰＬＰＥＧ−ＦＡをＰＢＳで洗い流したあと、共焦点レーザー顕微鏡にて、細胞を観察した。結果を図５ａ−ｄに示す。
図５ａ−ｄに示すとおり、ＢＮ−ＣＮＨ／ＰＬＰＥＧ(ａ)とＣＮＨ／ＰＬＰＥＧ(ｃ)は、ＫＢ細胞に取り込まれる量が少なかったが、ＢＮ−ＣＮＨ／ＰＬＰＥＧ―ＦＡ(ｂ)とＣＮＨ／ＰＬＰＥＧ―ＦＡ(ｄ)は、ＫＢ細胞に大量に取り込まれていた。(Selective uptake experiment of BN-CNH / PLPEG-FA of cancer cells)
A cell line (KB cells) derived from human oral epithelial cancer was cultured, and a BN-CNH / PLPEG-FA solution dispersed in PBS was added thereto to a final concentration of 10 μg / mL. After culturing for 24 hours, BN-CNH / PLPEG-FA that was not taken up by the cells was washed away with PBS, and then the cells were observed with a confocal laser microscope. The results are shown in FIGS. 5a-d.
As shown in FIGS. 5a-d, BN-CNH / PLPEG (a) and CNH / PLPEG (c) were less uptaken into KB cells, but BN-CNH / PLPEG-FA (b) and CNH / PLPEG -FA (d) was taken up in large quantities by KB cells.
また、ＢＮ−ＣＮＨ／ＰＬＰＥＧ−ＦＡのがん細胞による取り込み量を定量的に評価するため、細胞溶解試薬によって、細胞溶解液を作成し、この溶液中のタンパク質量とＣＮＨ量を測定した。タンパク質量は、ブラッドフォード法にて定量するため、遠心分離機(１８００ｘｇ，５０分)によって、細胞片を取り除き、また、ＣＮＨから分離した。上澄みの細胞溶解液(１０μＬ)に、ブラッドフォード液(２５０μＬ)を加えて、５９５ｎｍの波長での吸収強度を測定した。さらに、細胞が取り込んだＢＮ−ＣＮＨ量は、細胞溶解液をチップ型ソニケーションに１０分間かけた後、７００ｎｍの波長での吸収強度を測定することで濃度を見積もった。以上の操作によるタンパク質量とＣＮＨ量から、がん細胞でのＢＮ−ＣＮＨ／ＰＬＰＥＧ−ＦＡ取り込み量を算出した。結果を図５ｅに示す。
その結果、ＢＮ−ＣＮＨは、ＣＮＨと同様に、ＰＬＰＥＧ−ＦＡを修飾することによって、がん細胞に取り込まれやすくなることがわかった。Further, in order to quantitatively evaluate the amount of BN-CNH / PLPEG-FA taken up by cancer cells, a cell lysis solution was prepared with a cell lysis reagent, and the amount of protein and the amount of CNH in this solution were measured. In order to quantify the amount of protein by the Bradford method, cell debris was removed and separated from CNH by a centrifuge (1800 × g, 50 minutes). Bradford solution (250 μL) was added to the supernatant cell lysate (10 μL), and the absorption intensity at a wavelength of 595 nm was measured. Further, the amount of BN-CNH taken up by the cells was estimated by measuring the absorption intensity at a wavelength of 700 nm after applying the cell lysate to chip-type sonication for 10 minutes. The amount of BN-CNH / PLPEG-FA uptake in cancer cells was calculated from the amount of protein and the amount of CNH by the above operation. The result is shown in FIG.
As a result, it was found that BN-CNH can be easily taken up by cancer cells by modifying PLPEG-FA in the same manner as CNH.
［００３０］と［００３１］に記載と同様の実験を、正常細胞ＦＨｓ１７３Ｗｅを用いて行った。結果を図６ａ−ｅに示す。
図６ａ−eに示すとおり、ＦＡを付加したことによる細胞取り込み量に差は見られず、ＦＡがない場合と同様取り込み量は少なかった。ＢＮ−ＣＮＨ／ＰＬＰＥＧ(ａ)、ＢＮ−ＣＮＨ／ＰＬＰＥＧ−ＦＡ(ｂ)、ＣＮＨ／ＰＬＰＥＧ(ｃ)、ＣＮＨ/ＰＬＰＥＧ―ＦＡ(ｄ)のいずれの場合も、顕微鏡でみた細胞内取り込み量は少なく、また、これら試料の細胞内取り込み量測定結果にも優位な差は認められなかった(ｅ)。
上記［００３０］、［００３１］に記載の結果と合わせて考察することにより、ＢＮ−ＣＮＨ／ＰＬＰＥＧ−ＦＡは、がん細胞選択的に取り込まれるといえる。Experiments similar to those described in [0030] and [0031] were performed using normal cells FHs173We. The results are shown in FIGS. 6a-e.
As shown in FIGS. 6a-e, there was no difference in the amount of cellular uptake due to the addition of FA, and the amount of uptake was small as in the case without FA. In any case of BN-CNH / PLPEG (a), BN-CNH / PLPEG-FA (b), CNH / PLPEG (c), CNH / PLPEG-FA (d), the amount of cellular uptake observed with a microscope is small. In addition, no significant difference was observed in the measurement results of the amount of cellular uptake of these samples (e).
When considered together with the results described in [0030] and [0031] above, it can be said that BN-CNH / PLPEG-FA is selectively taken up by cancer cells.
本発明のＢＮ−ＣＮＨは、大量のホウ素化合物を内包および外壁に担持しており、特に、適切に表面修飾したＢＮ−ＣＮＨは、生体内投与した際に、腫瘍への蓄積を実現させ、ホウ素中性子捕捉療法の効果を高めることができるので、ホウ素中性子捕捉療法に有用な成分を提供する医薬製造業において利用できる。 The BN-CNH of the present invention carries a large amount of boron compound on the inner wall and the outer wall, and particularly, appropriately surface-modified BN-CNH realizes accumulation in a tumor when administered in vivo, and boron Since the effect of neutron capture therapy can be enhanced, it can be used in the pharmaceutical manufacturing industry that provides components useful for boron neutron capture therapy.
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