JP3826402B2 - Dispersion containing photocatalytic titanium dioxide composite fine particles - Google Patents
Dispersion containing photocatalytic titanium dioxide composite fine particles Download PDFInfo
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- JP3826402B2 JP3826402B2 JP2005276706A JP2005276706A JP3826402B2 JP 3826402 B2 JP3826402 B2 JP 3826402B2 JP 2005276706 A JP2005276706 A JP 2005276706A JP 2005276706 A JP2005276706 A JP 2005276706A JP 3826402 B2 JP3826402 B2 JP 3826402B2
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- titanium dioxide
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- photocatalytic titanium
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Description
本発明は、癌細胞、内分泌撹乱物質などに対する分子認識能を有する抗体などの生体分子を固定化し、紫外線の照射などによってこれらの分解作用を示す光触媒性二酸化チタン複合微粒子を含む分散液とその製造方法に関する。 Disclosed is a dispersion containing photocatalytic titanium dioxide composite fine particles in which biomolecules such as antibodies capable of recognizing molecules against cancer cells, endocrine disrupting substances and the like are immobilized, and these biocatalytic titanium dioxide composite particles exhibit a degrading action by irradiation with ultraviolet rays. Regarding the method.
近年、内分泌撹乱物質の分子認識能を有するＤＮＡなどの生体分子を支持体上に固定化した選択的吸着性を有する材料が環境浄化材料として提案されている（例えば、特許文献１参照）。一方、アナターゼ型二酸化チタンには光触媒作用があり、その強い酸化力により微生物、汚れ、悪臭物質等の有機物を分解することが知られている。現在では、二酸化チタンと活性炭やゼオライトなどの無機吸着剤を複合化することにより、二酸化チタンの分解効率を高めるような工夫がなされている（例えば特許文献２参照）。二酸化チタンの表面処理においても、パラジウムなどの還元反応促進触媒金属を二酸化チタン等の光触媒表面に析出させることで、光触媒の酸化、還元反応を促進することが考案されている（例えば、特許文献３参照）。 In recent years, a material having selective adsorptivity in which a biomolecule such as DNA having molecular recognition ability of an endocrine disrupting substance is immobilized on a support has been proposed as an environmental purification material (see, for example, Patent Document 1). On the other hand, anatase-type titanium dioxide has a photocatalytic action and is known to decompose organic substances such as microorganisms, dirt, and malodorous substances by its strong oxidizing power. At present, a device has been devised to increase the decomposition efficiency of titanium dioxide by combining titanium dioxide and an inorganic adsorbent such as activated carbon or zeolite (for example, see Patent Document 2). Also in the surface treatment of titanium dioxide, it has been devised to promote the oxidation and reduction reaction of the photocatalyst by precipitating a reduction reaction promoting catalyst metal such as palladium on the surface of the photocatalyst such as titanium dioxide (for example, Patent Document 3). reference).
しかしながら、ＤＮＡ等による内分泌攪乱物質の選択的吸着材料については、吸着した内分泌攪乱物質等の確実な除去・分解手段が無く、かつ吸着飽和の問題から浄化能力にも限界がある。また、前記の二酸化チタンの光触媒としての能力を高めようとする考案についても、特定物質の吸着や分解を指向していない。したがって、例えば内分泌攪乱物質のみを選択的に吸着し分解することは不可能である。このように、生体分子により特定の物質を選択的に吸着してこれを光触媒の強い酸化力によって分解する、すなわち「選択的吸着能と光触媒能との組み合わせ」については知られていない。
本発明は、選択的吸着能生体内での中性な生理的条件下においても安定して分散し、存在することが可能な、光触媒性二酸化チタン複合微粒子を提供することにある。 It is an object of the present invention to provide photocatalytic titanium dioxide composite fine particles that can stably disperse and exist even under neutral physiological conditions in vivo.
本発明者らは上記課題を解決するために鋭意検討を行い、二酸化チタン表面を親水性高分子で修飾した後に生体分子を固定化した光触媒性二酸化チタン複合微粒子が、選択的吸着能と光触媒能を両立できることを見い出し、本発明を完成した。 The inventors of the present invention have made extensive studies to solve the above-mentioned problems, and photocatalytic titanium dioxide composite fine particles in which biomolecules are immobilized after modifying the surface of titanium dioxide with a hydrophilic polymer have selective adsorption ability and photocatalytic ability. The present invention has been completed.
すなわち、本発明の光触媒性二酸化チタン複合微粒子を含む分散液はアナターゼ型二酸化チタン表面に親水性高分子アミンを有し、該親水性高分子のアミンと二酸化チタンはが強く結合しているとともに、前記親水性高分子のアミンに生体分子を固定化することを可能とし、さらに、分散剤等の他物質の添加無しに、分散性が極めて良好で安定な分散液である。 That is, the dispersion containing the photocatalytic titanium dioxide composite fine particles of the present invention has a hydrophilic polymer amine on the anatase-type titanium dioxide surface, and the hydrophilic polymer amine and titanium dioxide are strongly bonded together. A biomolecule can be immobilized on the hydrophilic polymer amine, and the dispersion is extremely stable and stable without adding other substances such as a dispersant.
本発明によれば、、選択的吸着能生体内での中性な生理的条件下においても安定して分散し、存在することができる。 According to the present invention, the selective adsorption ability can be stably dispersed and exist even under neutral physiological conditions in vivo.
本発明の実施の形態を図面に基づいて具体的に説明する。図１は本発明の光触媒性二酸化チタン複合微粒子を含む分散液を示す模式図である。本発明の光触媒性二酸化チタン複合微粒子を含む分散液はアナターゼ型二酸化チタン（１）と、生体分子と結合する親水性高分子アミン（２）を非プロトン性極性溶媒に分散させて、９０〜１８０℃で１〜１２時間水熱反応を行い、親水性高分子と二酸化チタンとの間で共有結合を生成させた後、水溶液に分散し、親水性高分子のアミンに生体分子（３）を固定化させたものである。これにより、本発明の光触媒性二酸化チタン複合微粒子のみで分散剤等の他物質の添加無しに、水溶液中において安定して分散可能であることを特徴とするものである。生体分子の固定化には、光触媒性二酸化チタン複合微粒子表面の二酸化チタンと親水性高分子の結合に関与していないフリーのアミンを用いることができる。生体分子側は、アミノ基、カルボキシル基、チオール基、糖鎖に由来するアルデヒド基等を持つ為、適切な架橋剤を用いることにより両者を共有結合させることが可能である。 Embodiments of the present invention will be specifically described with reference to the drawings. FIG. 1 is a schematic view showing a dispersion containing the photocatalytic titanium dioxide composite fine particles of the present invention. The dispersion containing the photocatalytic titanium dioxide composite fine particles of the present invention is obtained by dispersing anatase-type titanium dioxide (1) and a hydrophilic polymer amine (2) that binds to a biomolecule in an aprotic polar solvent, 90 to 180. Hydrothermal reaction for 1 to 12 hours at ℃ to generate a covalent bond between the hydrophilic polymer and titanium dioxide, then disperse in aqueous solution and fix biomolecule (3) to amine of hydrophilic polymer It has been made. Thus, the photocatalytic titanium dioxide composite fine particles of the present invention can be stably dispersed in an aqueous solution without adding other substances such as a dispersant. For immobilization of the biomolecule, free amine that is not involved in the binding of titanium dioxide on the surface of the photocatalytic titanium dioxide composite fine particle and the hydrophilic polymer can be used. Since the biomolecule side has an amino group, a carboxyl group, a thiol group, an aldehyde group derived from a sugar chain, etc., it is possible to covalently bond them together by using an appropriate crosslinking agent.
生体分子としては多種多様なものが考えられるが、最も利用されているものとしてタンパク質が挙げられる。本発明によれば、タンパク質として抗体、レセプターから低分子ペプチドまで好適に固定化が可能である。また、タンパク質の化学組成から光触媒性二酸化チタン複合微粒子への固定化にはアミノ基、カルボキシル基やチオール基、糖タンパクの場合ではアルデヒド基を固定化の際の標的官能基にすることが可能である。更には両者間をビオチンとアビジンの相互作用を利用して固定化することも可能である。 A wide variety of biomolecules can be considered, and proteins are most frequently used. According to the present invention, it is possible to suitably immobilize antibodies, receptors, and low molecular peptides as proteins. For immobilization from protein chemical composition to photocatalytic titanium dioxide composite fine particles, amino groups, carboxyl groups, thiol groups, and in the case of glycoproteins, aldehyde groups can be used as target functional groups for immobilization. is there. Furthermore, it is also possible to immobilize between the two using the interaction between biotin and avidin.
このような場合、ニ官能性のリンカー試薬を用いることで、光触媒性二酸化チタン複合微粒子と生体分子間の結合は達成される。ニ官能性のリンカー試薬としてホモ官能基のものを用いれば、光触媒性二酸化チタン複合微粒子表面のアミンと生体分子に由来するアミノ基間に容易に共有結合を導入することが可能である。また、ヘテロ官能基を持つものを用いれば、生体分子側にチオール基、カルボキシル基を持つ生体分子が導入可能である。 In such a case, the bond between the photocatalytic titanium dioxide composite fine particle and the biomolecule is achieved by using a bifunctional linker reagent. If a bifunctional linker reagent having a homofunctional group is used, it is possible to easily introduce a covalent bond between an amine on the surface of the photocatalytic titanium dioxide composite fine particle and an amino group derived from a biomolecule. In addition, if a compound having a heterofunctional group is used, a biomolecule having a thiol group or a carboxyl group on the biomolecule side can be introduced.
また、生体分子に限らず、蛍光色素や検出用プローブ物質等も適当な官能基の導入により光触媒性二酸化チタン複合微粒子への固定化が可能である。 Further, not only biomolecules but also fluorescent dyes and detection probe substances can be immobilized on the photocatalytic titanium dioxide composite fine particles by introducing appropriate functional groups.
アミノ基同士のホモリンカー試薬としては、Ｎ−ヒドロキシスクシンイミドエステルを持つもの、具体的にはＤｉｓｕｃｃｉｎｉｍｉｄｙｌ ｇｌｕｔａｒａｔｅやＢｉｓ（Ｓｕｌｆｏｓｕｃｃｉｎｉｍｉｄｙｌ） ｓｕｂｅｒａｔａｔｅ等およびイミドエステルを持つもの、具体的にはＤｉｍｅｔｈｙｌ ａｄｐｉｍｉｄａｔｅやＤｉｍｅｔｈｙｌ ｓｕｂｅｒｉｍｉｄａｔｅ等があり、好適に用いることができる。 Examples of homo-linker reagents between amino groups include those having N-hydroxysuccinimide ester, specifically, those having disuccinimidyl glutarate and Bis (sulfosuccinimidyl) sublate and imide ester, specifically, dimethymididimate and dimethylylate, etc. Can be preferably used.
また、ヘテロ官能基の組み合わせは、光触媒性二酸化チタン複合微粒子表面のアミンに対しては上述のＮ−ヒドロキシスクシンイミドエステル、イミドエステルが、生体物質側のチオール基に対しては、マレイミド基を有するもの、具体的にはＮ−（ε−Ｍａｌｅｉｍｉｄｏｃａｐｒｏｙｌｏｘｙ）ｓｕｃｃｉｎｉｍｉｄｅ ｅｓｔｅｒ等を用いることができる。 In addition, the combination of heterofunctional groups is the above-mentioned N-hydroxysuccinimide ester or imide ester for the amine on the surface of the photocatalytic titanium dioxide composite fine particle, and the maleimide group for the thiol group on the biological material side. Specifically, N- (ε-Maleimidecaproxy) succinimide ester or the like can be used.
核酸の固定化を行う場合にはポリメラーゼチェインリアクション（ＰＣＲ）によるＤＮＡ増幅の際に、アミノ化プライマー、チオール化プライマー、ビオチン化プライマーを用いて修飾ＤＮＡを合成することにより、同様の方法で光触媒性二酸化チタン複合微粒子へ固定化することが可能である。例えば、アミノ化ＤＮＡを固定化に用いる場合、光触媒性二酸化チタン複合微粒子表面のアミンとの間に二官能性ホモリンカーを用いれば、両者を混合するだけで固定化を行うことができる。また、チオール化ＤＮＡを用いる場合、上述のニ官能性ヘテロリンカーを用いれば、アミン−チオール間を結合することができる。
ビオチン化ＤＮＡを用いる場合には、光触媒性二酸化チタン複合微粒子にストレプトアビジンを導入することが必要であるが、この場合には、上述のアミノ基に対する二官能性ホモリンカーを用いることで容易に導入が可能である。
In the case of nucleic acid immobilization, photocatalytic property is synthesized in the same way by synthesizing modified DNA using aminated primer, thiolated primer and biotinylated primer during DNA amplification by polymerase chain reaction (PCR). It can be immobilized on titanium dioxide composite fine particles. For example, when aminated DNA is used for immobilization, if a bifunctional homolinker is used with the amine on the surface of the photocatalytic titanium dioxide composite fine particles, the immobilization can be performed only by mixing the two. Moreover, when using thiolated DNA, if an above-mentioned bifunctional heterolinker is used, between amine-thiol can be couple | bonded.
In the case of using biotinylated DNA, it is necessary to introduce streptavidin into the photocatalytic titanium dioxide composite fine particles. In this case, the introduction can be easily performed by using the above-mentioned bifunctional homolinker for the amino group. Is possible.
複合タンパク質や糖質などに由来する糖鎖の固定を行う場合は、シス−ジオールを過ヨウ素酸などによりアルデヒドに酸化し、光触媒性二酸化チタン複合微粒子のアミンとＳｏｄｉｕｍ ｃｙａｎｏｂｏｒｏｈｙｄｒｉｄｅの存在下でシッフ塩基を形成させることにより固定化が可能であるが、二官能性リンカーを用いて架橋形成することも可能である。 When immobilizing sugar chains derived from complex proteins or carbohydrates, cis-diol is oxidized to aldehyde with periodic acid or the like, and a Schiff base is formed in the presence of amines of photocatalytic titanium dioxide composite particles and sodium cyanoborohydride. Immobilization is possible by forming, but it is also possible to form a crosslink using a bifunctional linker.
タンパク質や糖質の一部など、生体分子側がカルボキシル基を持つ場合、１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド（ＥＤＣ）により活性化を行い、光触媒性二酸化チタン複合微粒子と混合することにより両者間の架橋が可能である。 When the biomolecule side has a carboxyl group, such as a part of protein or carbohydrate, it is activated with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) and mixed with photocatalytic titanium dioxide composite fine particles Thus, cross-linking between the two is possible.
本発明で用いる親水性高分子としては水溶液中での分散性の観点から水溶性高分子が望ましい。水溶性高分子としては、重量平均分子量が１０００〜１０００００の範囲にあるアミンであればいずれも使用可能であるが、例えばポリアミノ酸、ポリペプチド、ポリアミン類、およびアミン単位を有する共重合体（コポリマー）などが挙げられる。具体的には、水溶性高分子の加水分解性および溶解度の観点から、ポリエチレンイミン、ポリビニルアミン、ポリアリルアミン等のポリアミン類がより好適に使用される。 The hydrophilic polymer used in the present invention is preferably a water-soluble polymer from the viewpoint of dispersibility in an aqueous solution. As the water-soluble polymer, any amine having a weight average molecular weight in the range of 1,000 to 100,000 can be used. For example, polyamino acids, polypeptides, polyamines, and copolymers having amine units (copolymers). ) And the like. Specifically, polyamines such as polyethyleneimine, polyvinylamine, and polyallylamine are more preferably used from the viewpoint of hydrolyzability and solubility of the water-soluble polymer.
また、本発明の光触媒性二酸化チタン複合微粒子の材料として用いる二酸化チタンとしては、癌治療用として体内への適用の場合など、その使用形態の自由度の観点から分散粒経が２〜２００ｎｍであることが望ましい。さらに、本発明で用いる二酸化チタンとしては、光触媒活性の観点からアナターゼ型であることが望ましい。 In addition, the titanium dioxide used as the material of the photocatalytic titanium dioxide composite fine particles of the present invention has a dispersed particle size of 2 to 200 nm from the viewpoint of the degree of freedom of its usage, such as when applied to the body for cancer treatment. It is desirable. Furthermore, the titanium dioxide used in the present invention is desirably an anatase type from the viewpoint of photocatalytic activity.
また、本発明の光触媒性二酸化チタン複合微粒子は、分散性の観点から分散粒経が２〜５００ｎｍであることが好ましい。癌治療用として体内への適用の場合、腫瘍細胞への蓄積効果の観点から分散粒経が５０〜２００ｎｍであることがより好ましい。このような範囲とすることで、生理的条件下で２４時間以上にわたって、安定した分散が可能となる。尚、ここでいう分散粒径とは、動的光散乱法によって測定を行い、キュムラント法解析から算出される平均値のことを示している。また、ここでいう生理的条件下とは２５℃、１気圧で
（１３７ｍＭ ＮａＣｌ，８．１ｍＭ Ｎａ２ＨＰＯ４，２．６８ｍＭ ＫＣｌ，１．４７ｍＭ ＫＨ２ＰＯ４）の組成であるリン酸緩衝食塩水（ｐＨ７．４）存在下のことを示す。
The photocatalytic titanium dioxide composite fine particles of the present invention preferably have a dispersed particle size of 2 to 500 nm from the viewpoint of dispersibility. In the case of application to the body for cancer treatment, the dispersed particle size is more preferably 50 to 200 nm from the viewpoint of the effect of accumulation in tumor cells. By setting it as such a range, stable dispersion | distribution is attained over 24 hours or more on physiological conditions. In addition, the dispersion | distribution particle size here shows the average value measured by the dynamic light-scattering method and computed from cumulant method analysis. The physiological condition referred to here is a phosphate buffered saline (pH 7.4) having a composition of (137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4) at 25 ° C. and 1 atm. Indicates presence.
さらに、上述した二酸化チタンが少なくとも表面に存在すれば、たとえば磁性粒子と二酸化チタンとの複合体のようなものであっても、水溶液中での特性は近似し、親水性高分子アミンにより表面を修飾することで、アミンを介した生体分子の固定化は可能であるため、同様の製造法、精製法を適用することができる。 Furthermore, if the above-described titanium dioxide is present at least on the surface, even in the case of, for example, a composite of magnetic particles and titanium dioxide, the characteristics in an aqueous solution are approximated, and the surface is covered with a hydrophilic polymer amine. By modifying, biomolecules can be immobilized via amines, and the same production method and purification method can be applied.
また、本発明の光触媒性二酸化チタン複合微粒子を含む分散液における光触媒性二酸化チタン複合微粒子は、表面上に存在するアミンにより、粒子間に電気的斥力が働くために凝集することなく、少なくとも２４時間以上にわたって安定に存在することによる。しかも、基本的にｐＨの変動や無機塩類の添加に対しても極めて安定である。さらに、表面上に存在するアミンによって表面電荷が正に帯電していることから、一般的に負の表面電荷をもつ細胞への親和性、取込み性が著しく高く、癌細胞の破壊を目的とした医療用材料として極めて有用である。これらの観点から、表面電位の最適範囲としては、良好な分散性と細胞取込み性を達成できる範囲にあればよく、＋２０ｍＶ以上あればよい。さらに望ましくは、一般に自主分散（粒子が沈殿しない状態）が十分に達成できる電位として＋４０ｍＶ以上あればよい。 Further, the photocatalytic titanium dioxide composite fine particles in the dispersion containing the photocatalytic titanium dioxide composite fine particles of the present invention are at least 24 hours without agglomeration due to an electrical repulsive force between the particles due to the amine present on the surface. It is because it exists stably over the above. Moreover, it is basically very stable against pH fluctuations and addition of inorganic salts. Furthermore, since the surface charge is positively charged by the amine present on the surface, in general, the affinity to and uptake of cells with a negative surface charge is remarkably high, and the purpose is to destroy cancer cells. It is extremely useful as a medical material. From these viewpoints, the optimum range of the surface potential may be in a range in which good dispersibility and cell uptake can be achieved, and may be +20 mV or more. More preferably, it is generally sufficient that the electric potential at which sufficient self-dispersion (state in which particles do not precipitate) can be sufficiently achieved is +40 mV or more.
また、本発明の光触媒性二酸化チタン複合微粒子を含む分散液における光触媒性二酸化チタン複合微粒子は、光触媒性二酸化チタン複合微粒子間に電気的斥力が働くために凝集することなく、少なくとも２４時間以上にわたって安定することができ、これを達成できる塩濃度の範囲として、１Ｍ以下であればよい。さらに望ましくは、生体への適用を考えた場合に生体内での中性な生理的条件下においても安定して分散し、存在することができればよく、塩濃度として１００ｍＭ〜３００ｍＭ程度であればよい。 In addition, the photocatalytic titanium dioxide composite fine particles in the dispersion containing the photocatalytic titanium dioxide composite fine particles of the present invention are stable for at least 24 hours without agglomeration due to the electric repulsion between the photocatalytic titanium dioxide composite fine particles. The range of the salt concentration that can be achieved is 1M or less. More desirably, it is only necessary to stably disperse and exist even under neutral physiological conditions in the living body when considering application to a living body, and the salt concentration may be about 100 mM to 300 mM. .
また、本発明の光触媒性二酸化チタン複合微粒子を含む分散液における光触媒性二酸化チタン複合微粒子は、光触媒性二酸化チタン複合微粒子間に電気的斥力が働くために凝集することなく、少なくとも２４時間以上にわたって安定することができ、これを達成できる光触媒性二酸化チタン粒子濃度の範囲として、重量百分率で２０％以下であればよい。さらに望ましくは、生体への適用を考えた場合に細胞に対する安全性の観点から、重量百分率で０．１％〜０．０００１％であればよい。 In addition, the photocatalytic titanium dioxide composite fine particles in the dispersion containing the photocatalytic titanium dioxide composite fine particles of the present invention are stable for at least 24 hours without agglomeration due to the electric repulsion between the photocatalytic titanium dioxide composite fine particles. The range of the photocatalytic titanium dioxide particle concentration that can be achieved is 20% or less in terms of weight percentage. More desirably, it may be 0.1% to 0.0001% in terms of weight percentage from the viewpoint of safety with respect to cells when considering application to a living body.
以上のことから、本発明の光触媒性二酸化チタン複合微粒子を含む分散液を水、種々のｐＨ緩衝液、輸液、あるいは生理食塩水を用いた、均一で安定な分散液として提供することが可能となる。また、本分散液を含む軟膏やスプレー剤等も製造が可能である。この特性は、特に二酸化チタンを体内外のＤＤＳに応用する際に極めて有用である。すなわち、本発明の光触媒性二酸化チタン複合微粒子を含む分散液は中性付近の生理的条件においても凝集することがないために、患部組織に直接注射したり静脈に注射してターゲティングを行うことが可能となる。また、本分散液を含む軟膏やスプレー剤を皮膚等の患部に直接塗布し、太陽光や紫外線ランプ等により治療を施すことが可能となる。 From the above, it is possible to provide a dispersion containing the photocatalytic titanium dioxide composite fine particles of the present invention as a uniform and stable dispersion using water, various pH buffer solutions, infusion solutions, or physiological saline. Become. In addition, ointments and sprays containing this dispersion can also be produced. This characteristic is extremely useful particularly when titanium dioxide is applied to DDS inside and outside the body. That is, since the dispersion containing the photocatalytic titanium dioxide composite fine particles of the present invention does not aggregate even under physiological conditions near neutrality, it can be directly injected into the affected tissue or injected into the vein for targeting. It becomes possible. In addition, an ointment or spray containing this dispersion can be applied directly to the affected area such as the skin and treated with sunlight or an ultraviolet lamp.
本発明の光触媒性二酸化チタン複合微粒子を含む分散液における光触媒性二酸化チタン複合微粒子を励起、活性化させるための光源装置は特別である必要はないが、二酸化チタンのバンドギャップの関係上その波長は４００ｎｍ以下であることが望ましい。皮膚等の外用用途では、太陽光や通常の紫外線ランプ、あるいはブラックライトを好適に使用できる。また、体内の患部に対しては内視鏡に紫外線ファイバーを装着することにより紫外線を照射すれば良い。さらに、特に２８０ｎｍ付近の紫外線を局所的に患部に照射して病変部を破壊しようとする光療法を想定した場合では、その作用増強剤として本発明の光触媒性二酸化チタン複合微粒子を含む分散液を適用することも可能である。 The light source device for exciting and activating the photocatalytic titanium dioxide composite fine particles in the dispersion containing the photocatalytic titanium dioxide composite fine particles of the present invention does not need to be special, but the wavelength is related to the band gap of titanium dioxide. It is desirable that it is 400 nm or less. For external use such as skin, sunlight, a normal ultraviolet lamp, or black light can be suitably used. Moreover, what is necessary is just to irradiate an ultraviolet-ray by attaching an ultraviolet fiber to an endoscope with respect to the affected part in a body. Furthermore, in particular, when assuming phototherapy for locally irradiating the affected area with ultraviolet rays of around 280 nm to destroy the affected area, a dispersion containing the photocatalytic titanium dioxide composite fine particles of the present invention is used as its action enhancer. It is also possible to apply.
さらに、本発明の光触媒性二酸化チタン複合微粒子を含む分散液における光触媒性二酸化チタン複合微粒子は、表面上に存在するアミンによって表面電荷が正に帯電していることから、一般的に負の表面電荷をもつ細胞への親和性、取込み性が著しく高く、本発明の光触媒性二酸化チタン複合微粒子と細胞とが接触すると直ちに細胞への結合や取込みが始まる。このことから、特に生体の皮膚表面や、あるいは気管、消化器などのあるいは生体内部の表層部や、生体内に存在する様々な患部への適用が非常に有効であり、さらに、生体分子を固定化させることで癌細胞内での局在化を可能にする。例えば、核内への移行シグナルを結合することで、核内DNAへの接近を容易にし、より高い治療効果を得ることができる。例えば、本発明の光触媒性二酸化チタン微粒子を含む分散液を含む軟膏やスプレー剤を皮膚癌や喉頭癌といった癌患部に直接塗布したり、あるいは注射により固形ガンに局所投与した後に、太陽光や紫外線ランプ等により治療を施すことにより簡便でかつ高い治療効果を得ることができるものである。 Furthermore, the photocatalytic titanium dioxide composite fine particles in the dispersion containing the photocatalytic titanium dioxide composite fine particles of the present invention generally have a negative surface charge because the surface charge is positively charged by the amine present on the surface. The affinity and the uptake | capture property to the cell which has, and remarkably high are taken, and the coupling | bonding and uptake | capture to a cell will start as soon as the photocatalytic titanium dioxide composite microparticle of this invention and a cell contact. For this reason, it is particularly effective for application to the skin surface of the living body, the surface layer of the trachea, digestive organs, etc. or inside the living body, and various affected parts existing in the living body. Allows localization in cancer cells. For example, by binding a signal for translocation into the nucleus, access to the nuclear DNA can be facilitated, and a higher therapeutic effect can be obtained. For example, after applying an ointment or spray containing the dispersion containing the photocatalytic titanium dioxide fine particles of the present invention directly to a cancer affected area such as skin cancer or laryngeal cancer, or locally administering to a solid cancer by injection, sunlight or ultraviolet rays By performing treatment with a lamp or the like, a simple and high therapeutic effect can be obtained.
以下、本発明を実施例に従って詳細に説明する。ただし、本発明はこの実施例に制限されるものではない。 Hereinafter, the present invention will be described in detail according to examples. However, the present invention is not limited to this embodiment.
（実施例１）
二酸化チタンへのポリエチレンイミンの導入
チタンテトライソプロポキシド３．６ｇとイソプロパノール３．６ｇを混合し、氷冷下で６０ｍｌの超純水に滴下して加水分解を行った。滴下後に室温で３０分間攪拌した。攪拌後、１２Ｎ硝酸１ｍｌを滴下して８０℃で８時間攪拌を行い、ペプチゼーションした。ペプチゼーション終了後０．４５μｍのフィルターで濾過し、さらに脱塩カラムＰＤ−１０（アマシャム・ファルマシア・バイオサイエンス社製）を用いて溶液交換して固形成分１％の酸性二酸化チタンゾルを調製した。この分散液を１００ｍｌ容のバイアル瓶に入れ、２００Ｈｚで３０分間超音波処理を行った。超音波処理を行う前後の平均分散粒経はそれぞれ、３６．４ｎｍ、２０．２ｎｍであった。超音波処理後、溶液を濃縮して固形成分２０％の二酸化チタンゾルを調製した。得られた二酸化チタンゾル０．７５ｍｌを２０ｍｌのジメチルホルムアミド(ＤＭＦ)に分散させ、ポリエチレンイミン（平均分子量：１００００、和光純薬社製）４５０ｍｇを溶解したＤＭＦ１０ｍｌを添加後、攪拌して混合した。水熱反応容器（ＨＵ−５０、三愛科学社製）に溶液を移し変え、１５０℃で６時間合成を行った。反応終了後、反応容器温度が５０℃以下になるまで冷却し、２倍量のイソプロパノールを添加し、ポリエチレンイミン結合二酸化チタン微粒子を沈殿させ、遠心後に上清を除去することにより未反応のポリエチレンイミンを分離した。７０％エタノールを添加して洗浄を行い、遠心後にエタノールを除去した。蒸留水を１０ｍｌ添加後、２００Ｈｚで３０分間超音波処理を行い、ポリエチレンイミン結合二酸化チタン微粒子を分散させた。超音波処理後、０．４５μｍのフィルターで濾過して、固形成分１．５％のポリエチレンイミン結合二酸化チタン微粒子の分散液を得た。作製したポリエチレンイミン結合二酸化チタン微粒子の分散粒径を、ゼータサイザーナノＺＳ（シスメックス社製）を用いて、ゼータ電位測定セルにポリエチレンイミン結合二酸化チタン微粒子の分散液０．7５ｍｌを仕込み、溶媒の各種パラメーターを水と同値に設定し、２５℃にて動的光散乱法により測定したところ、ポリエチレンイミン結合二酸化チタン微粒子の平均粒径は６７．７ｎｍであった。
Example 1
Introduction of Polyethyleneimine into Titanium Dioxide 3.6 g of titanium tetraisopropoxide and 3.6 g of isopropanol were mixed and hydrolyzed by adding dropwise to 60 ml of ultrapure water under ice cooling. After dropping, the mixture was stirred at room temperature for 30 minutes. After stirring, 1 ml of 12N nitric acid was added dropwise, and the mixture was stirred at 80 ° C. for 8 hours for peptization. After completion of peptization, the solution was filtered with a 0.45 μm filter, and the solution was exchanged using a desalting column PD-10 (Amersham Pharmacia Bioscience) to prepare an acidic titanium dioxide sol having a solid content of 1%. This dispersion was placed in a 100 ml vial and sonicated at 200 Hz for 30 minutes. The average dispersed particle sizes before and after the ultrasonic treatment were 36.4 nm and 20.2 nm, respectively. After sonication, the solution was concentrated to prepare a titanium dioxide sol having a solid content of 20%. 0.75 ml of the obtained titanium dioxide sol was dispersed in 20 ml of dimethylformamide (DMF), and 10 ml of DMF in which 450 mg of polyethyleneimine (average molecular weight: 10,000, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved was added and then stirred and mixed. The solution was transferred to a hydrothermal reaction vessel (HU-50, Sanai Kagaku Co., Ltd.), and synthesis was performed at 150 ° C. for 6 hours. After completion of the reaction, the reaction vessel is cooled until the temperature reaches 50 ° C. or lower, twice the amount of isopropanol is added, polyethyleneimine-bound titanium dioxide fine particles are precipitated, and the supernatant is removed after centrifugation to remove unreacted polyethyleneimine. Separated. Washing was performed by adding 70% ethanol, and ethanol was removed after centrifugation. After adding 10 ml of distilled water, ultrasonic treatment was performed at 200 Hz for 30 minutes to disperse the polyethyleneimine-bound titanium dioxide fine particles. After ultrasonic treatment, the mixture was filtered through a 0.45 μm filter to obtain a dispersion of polyethyleneimine-bound titanium dioxide fine particles having a solid content of 1.5%. Using the Zeta Sizer Nano ZS (manufactured by Sysmex Corporation), 0.75 ml of a dispersion of polyethylene imine-bonded titanium dioxide fine particles was charged into a zeta potential measurement cell using the prepared polyethyleneimine-bonded titanium dioxide fine particles. When the parameter was set to the same value as water and measured by a dynamic light scattering method at 25 ° C., the average particle diameter of the polyethyleneimine-bound titanium dioxide fine particles was 67.7 nm.
（実施例２）
ポリエチレンイミン結合二酸化チタン微粒子へのタンパク質の固定化
０．１ｍgのストレプトアビジン（ピアース社製）を含む５０ｍＭの２[４−（２−ヒドロキシエチル）−１−ピペラジニル]エタンスルホン酸（ＨＥＰＥＳ）緩衝液（ｐＨ８．０）１ｍｌに２０ｍＭの１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド（ＥＤＣ）と５ｍＭのＮ−ヒドロキシこはく酸イミド（ＮＨＳ）の混合液０．１ｍｌを添加して５分間攪拌を行い、ストレプトアビジンのもつカルボキシル基を活性化した。攪拌終了後、１０ｍＭ酢酸緩衝液（ｐＨ５．０）で平衡化した脱塩カラムＮＡＰ−１０（アマシャム・ファルマシア・バイオサイエンス社製）を用いてゲル濾過を行い、未反応のＥＤＣおよびＮＨＳを除去した。ストレプトアビジンを含む溶液０．１ｍｌを実施例１で得られたポリエチレンイミン結合二酸化チタン微粒子を含む分散液２ｍｌに添加し、１０分間、４℃にて穏やかに攪拌した。この溶液を透析チューブ(分画分子量：１０００００、ピアース社製)に移し、２０ｍＭトリスヒドロキシメチルアミノメタン−塩酸緩衝液（ｐＨ８．０）に対して１２時間透析を行った。透析チューブ内の溶液を回収し、２倍量のイソプロパノールを添加し、１０分間、４０００ｇにて遠心分離し、沈殿を７０％エタノールで洗浄後、１００ｍＭリン酸緩衝食塩水（ｐＨ７．５、日本ジーン社製）１ｍｌに溶解した。これにより、ストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子の分散液を得た。この分散液の粒径を、ゼータサイザーナノＺＳ（シスメックス社製）を用いて、ゼータ電位測定セルにこの分散液０．7５ｍｌを仕込み、溶媒の各種パラメーターを水と同値に設定し、２５℃にて動的光散乱法により測定したところ、作製した光触媒性二酸化チタン複合微粒子の平均粒径は６８．２ｎｍであった。
(Example 2)
Immobilization of protein on polyethyleneimine-bound
（実施例３）
ストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子の生体分子機能性の確認
実施例２で得られたストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子の分散液０．１ｍｌに対し、それぞれ１ｍＭから１００ｎＭまで１０倍刻みで希釈した異なる濃度のビオチンダイマー（ＥＺ−Ｌｉｎｋ ＰＥＯ−Ｂｉｏｔｉｎ Ｄｉｍｅｒ、ピアース社製）を０．０１ｍｌ添加し、３７℃にて１０分間静置し、５９５ｎｍの吸光度をマイクロタイタープレートリーダー（Ｂｅｎｃｈ Ｍａｒｋ、バイオラッド社製）にて測定した。結果を図２に示す。明らかにビオチンダイマーの濃度に応じて溶液の濁度が上昇しており、光触媒性二酸化チタン複合微粒子にストレプトアビジンが効率的に固定化されていることが判明した。
Example 3
Confirmation of Biomolecular Functionality of Photocatalytic Titanium Dioxide Composite Fine Particles Immobilized with Streptavidin From 1 mM each to 0.1 ml of the dispersion of photocatalytic titanium dioxide composite fine particles immobilized with streptavidin obtained in Example 2 Add 0.01 ml of different concentrations of biotin dimer (EZ-Link PEO-Biotin Dimer, Pierce) diluted to 100 nM in 10-fold increments, leave it at 37 ° C. for 10 minutes, and absorb the absorbance at 595 nm in a microtiter plate. Measurement was performed with a reader (Bench Mark, manufactured by Bio-Rad). The results are shown in FIG. Obviously, the turbidity of the solution increased according to the concentration of biotin dimer, and it was found that streptavidin was efficiently immobilized on the photocatalytic titanium dioxide composite fine particles.
（実施例４）
蛍光色素標識を行った光触媒性二酸化チタン複合微粒子の作製
実施例１で得られたポリエチレンイミン結合二酸化チタン微粒子の分散液５００μｌを１００ｍＭリン酸緩衝食塩水（ｐＨ７．５）で平衡化した脱塩カラムＮＡＰ−１０（アマシャム・ファルマシア・バイオサイエンス社製）を用いてゲル濾過を行って溶液交換し、そこへ、最終濃度が０．８ｍＭになるようにＤＭＳＯに溶解したフルオレセインイソチオシアネート（ピアース社製）を加え、３０分間室温で穏やかに攪拌した。反応終了後、あらかじめ蒸留水にて平衡化したＰＤ−１０（アマシャム・ファルマシア・バイオサイエンス社製）により脱塩を行い、その後、溶液を２ｍｌにまで濃縮した。この蛍光色素標識を行った光触媒性二酸化チタン複合微粒子を含む分散液の液滴をガラスプレート上にのせ、蛍光顕微鏡で観察したところ、液滴と空気の境界面で明確に差が見られるようなフルオレセインに起因する蛍光が観察された（図３）。
Example 4
Preparation of Photocatalytic Titanium Dioxide Composite Fine Particles Labeled with Fluorescent Dye Desalting column obtained by equilibrating 500 μl of the dispersion of polyethyleneimine-bound titanium dioxide fine particles obtained in Example 1 with 100 mM phosphate buffered saline (pH 7.5) Gel filtration was performed using NAP-10 (manufactured by Amersham Pharmacia Bioscience) to exchange the solution, and fluorescein isothiocyanate (manufactured by Pierce) dissolved in DMSO so that the final concentration was 0.8 mM. And gently stirred at room temperature for 30 minutes. After completion of the reaction, desalting was performed with PD-10 (manufactured by Amersham Pharmacia Bioscience) previously equilibrated with distilled water, and then the solution was concentrated to 2 ml. Droplets of dispersion liquid containing photocatalytic titanium dioxide composite fine particles labeled with this fluorescent dye are placed on a glass plate and observed with a fluorescence microscope. As a result, a clear difference can be seen at the interface between the droplets and air. Fluorescence due to fluorescein was observed (FIG. 3).
（実施例５）
光触媒性二酸化チタン複合微粒子の細胞取込み性の評価 実施例１で得られた二酸化チタンゾル０．７５ｍｌを２０ｍｌのジメチルホルムアミド（ＤＭＦ）に分散させ、ポリアクリル酸（平均分子量：５０００、和光純薬社製）０．２ｇを溶解したＤＭＦを１０ｍｌ添加後、攪拌して混合した。水熱反応容器に溶液を移し変え、１８０℃で６時間水熱合成を行った。反応終了後、反応容器温度が５０℃以下になるまで冷却し、溶液を取り出した後に水８０ｍｌを添加して攪拌混合した。エバポレータでＤＭＦおよび水を除去した後に、再度、水２０ｍｌを添加してポリアクリル酸結合二酸化チタン微粒子の水溶液とした。２Ｎ塩酸１ｍｌを添加して二酸化チタン粒子を沈殿させて、遠心後に上清を除去することにより未反応のポリアクリル酸を分離した。再度水を添加して洗浄を行い、遠心後に水を除去した。５０ｍＭリン酸緩衝液（ｐＨ７．０）を１０ｍｌ添加後、２００Ｈｚで３０分間超音波処理を行い、二酸化チタン粒子を分散させた。超音波処理後、０．４５μｍのフィルターで濾過し、重量百分率で０．２５％のポリアクリル酸結合二酸化チタン微粒子の分散液を得た。ゼータサイザーナノＺＳ（シスメックス社製）を用いて、ゼータ電位測定セルにポリエチレンイミン結合二酸化チタン微粒子の分散液０．７５ｍｌを仕込み、溶媒の各種パラメーターを水と同値に設定し、２５℃にて動的光散乱法により測定したところ、作製したポリアクリル酸結合二酸化チタン微粒子の平均粒径は４５．９ｎｍであった。このポリアクリル酸結合二酸化チタン微粒子の分散液２ｍｌに対して、０．８Ｍ １−Ｅｔｈｙｌ−３−［３−Ｄｉｍｅｔｈｙｌａｍｉｎｏｐｒｏｐｙｌ］Ｃａｒｂｏｄｉｉｍｉｄｅ Ｈｙｄｒｏｃｈｌｏｒｉｄｅを２５０μｌおよびＮ−Ｈｙｄｒｏｘｙｓｕｃｃｉｎｉｍｉｄｅを２５０μｌ加えて、撹拌しながら室温で１時間反応させた。１０ｍＭ 酢酸緩衝液（ｐＨ５．０）で平衡化した脱塩カラムＮＡＰ−１０（アマシャム・ファルマシア・バイオサイエンス社製）を用いてゲル濾過を行って溶液交換し、その後に１０ｍＭ酢酸緩衝液（ｐＨ５．０）を用いて全量を９．５ｍｌとした。そこへ、ＤＭＦに溶解させた１００ｍＭ ５−ａｍｉｎｏ ｆｌｕｏｒｅｓｃｅｉｎ（ＮＣＩ社製）を５μｌ加え、遮光下で撹拌しながら室温で１時間反応させた。次に、０．１Ｍのエタノールアミン（和光純薬工業社製）水溶液を５００μｌ加え、遮光下で撹拌しながら室温で３０分間反応させた。この溶液を１００ｍＭリン酸緩衝食塩水（ｐＨ７．５）で平衡化した脱塩カラムＰＤ−１０を用いてゲル濾過を行って溶液交換し、未反応の５−ａｍｉｎｏ ｆｌｕｏｒｅｓｃｅｉｎを分離し、その後、溶液を２ｍｌにまで濃縮した。これを蛍光色素標識ポリアクリル酸結合二酸化チタン複合微粒子の分散液とした。
また、実施例１で得られたポリエチレンイミン結合二酸化チタン微粒子の分散液を５００μｌを、１００ｍＭリン酸緩衝食塩水（ｐＨ７．５）で平衡化した脱塩カラムＮＡＰ−１０を用いてゲル濾過を行って溶液交換し、そこへ、最終濃度が０．８ｍＭになるようにＤＭＳＯに溶解したフルオレセインイソチオシアネート（ピアース社製）を加え、３０分間室温で穏やかに攪拌した。反応終了後、あらかじめＰＢＳにて平衡化したＰＤ−１０（アマシャム・ファルマシア・バイオサイエンス社製）により溶液交換を行い、その後、溶液を２ｍｌにまで濃縮した。これを蛍光色素標識光触媒性二酸化チタン複合微粒子の分散液とした。
次に、メラノーマ細胞株Ｔ−２４を１０％血清を含むＦ１２培地（ギブコ社製）で１００％コンフルエントにになるまで培養し、フラスコを１００ｍＭリン酸緩衝食塩水（ｐＨ７．４）で２回洗浄し、１００ｍＭトリプシン−エチレンジアミン三酢酸溶液を１ｍｌ添加し、１０分静置後、フラスコ壁面より剥離した細胞を回収し、９ｍｌの１０％血清を含むＦ１２培地で希釈した。細胞数を血球計算盤により計測し、５×１０４個の細胞を含む培地５００μｌをそれぞれ２４穴マイクロタイタープレートに接種し、最終濃度０．０１％になるように分注した。そこに、先の蛍光色素標識ポリアクリル酸結合二酸化チタン複合微粒子の分散液および蛍光色素標識光触媒性二酸化チタン複合微粒子の分散液をそれぞれ最終濃度０．０１％となるよう１００μｌ加え、２４時間ＣＯ２インキュベータ内で培養した。その後、細胞のフラスコへの接着を確認し、フラスコを１００ｍＭリン酸緩衝食塩水にて洗浄し、２００μｌの１０％血清を含むＦ１２培地を添加し、蛍光顕微鏡により観察を行った（図４）。蛍光視野像を観察した結果、蛍光色素標識ポリアクリル酸結合二酸化チタン複合微粒子よりも蛍光色素標識ポ光触媒性二酸化チタン複合微粒子のほうが明らかに細胞に対して高い親和性と細胞取込み性をもつことが確認された。
(Example 5)
Evaluation of cell uptake of photocatalytic titanium dioxide composite fine particles 0.75 ml of the titanium dioxide sol obtained in Example 1 was dispersed in 20 ml of dimethylformamide (DMF), and polyacrylic acid (average molecular weight: 5000, manufactured by Wako Pure Chemical Industries, Ltd.) ) After adding 10 ml of DMF in which 0.2 g was dissolved, the mixture was stirred and mixed. The solution was transferred to a hydrothermal reaction vessel, and hydrothermal synthesis was performed at 180 ° C. for 6 hours. After completion of the reaction, the reaction vessel was cooled to 50 ° C. or lower, and after taking out the solution, 80 ml of water was added and stirred and mixed. After removing DMF and water with an evaporator, 20 ml of water was added again to obtain an aqueous solution of polyacrylic acid-bonded titanium dioxide fine particles. Titanium dioxide particles were precipitated by adding 1 ml of 2N hydrochloric acid, and unreacted polyacrylic acid was separated by removing the supernatant after centrifugation. Water was added again for washing, and water was removed after centrifugation. After adding 10 ml of 50 mM phosphate buffer (pH 7.0), ultrasonic treatment was performed at 200 Hz for 30 minutes to disperse the titanium dioxide particles. After ultrasonic treatment, the mixture was filtered through a 0.45 μm filter to obtain a dispersion of polyacrylic acid-bonded titanium dioxide fine particles having a weight percentage of 0.25%. Using Zeta Sizer Nano ZS (manufactured by Sysmex Corporation), 0.75 ml of a dispersion of polyethyleneimine-bonded titanium dioxide fine particles was charged into a zeta potential measurement cell, and various parameters of the solvent were set to the same values as water and operated at 25 ° C. The average particle size of the prepared polyacrylic acid-bonded titanium dioxide fine particles was 45.9 nm as measured by a dynamic light scattering method. To 2 ml of the dispersion of the polyacrylic acid-bonded titanium dioxide fine particles, 250 μl of 0.8M 1-Ethyl-3- [3-dimethylaminopropyl] carbohydrate Hydrochloride and 250 μl of N-hydroxysuccinimide were added and stirred at room temperature for 1 hour. Reacted. Gel filtration was performed using a desalting column NAP-10 (manufactured by Amersham Pharmacia Bioscience) equilibrated with 10 mM acetate buffer (pH 5.0), and then the solution was exchanged. Thereafter, 10 mM acetate buffer (pH 5. 0) was used to bring the total volume to 9.5 ml. Thereto, 5 μl of 100 mM 5-aminofluorescein (manufactured by NCI) dissolved in DMF was added and allowed to react at room temperature for 1 hour with stirring under light shielding. Next, 500 μl of 0.1 M ethanolamine (manufactured by Wako Pure Chemical Industries, Ltd.) aqueous solution was added and allowed to react at room temperature for 30 minutes while stirring under light shielding. This solution was subjected to gel filtration using a desalting column PD-10 equilibrated with 100 mM phosphate buffered saline (pH 7.5) to exchange the solution, and unreacted 5-aminofluorescein was separated. Was concentrated to 2 ml. This was used as a dispersion of fluorescent dye-labeled polyacrylic acid-bonded titanium dioxide composite fine particles.
Further, gel filtration was performed using a desalting column NAP-10 in which 500 μl of the dispersion of polyethyleneimine-bound titanium dioxide fine particles obtained in Example 1 was equilibrated with 100 mM phosphate buffered saline (pH 7.5). Then, fluorescein isothiocyanate (manufactured by Pierce) dissolved in DMSO so as to have a final concentration of 0.8 mM was added thereto, and gently stirred at room temperature for 30 minutes. After completion of the reaction, the solution was exchanged with PD-10 (manufactured by Amersham Pharmacia Bioscience) previously equilibrated with PBS, and then the solution was concentrated to 2 ml. This was used as a dispersion of fluorescent dye-labeled photocatalytic titanium dioxide composite fine particles.
Next, the melanoma cell line T-24 is cultured in F12 medium (manufactured by Gibco) containing 10% serum until it becomes 100% confluent, and the flask is washed twice with 100 mM phosphate buffered saline (pH 7.4). Then, 1 ml of a 100 mM trypsin-ethylenediamine triacetic acid solution was added, and after standing for 10 minutes, the detached cells were collected from the flask wall and diluted with 9 ml of F12 medium containing 10% serum. The number of cells was counted with a hemocytometer, 500 μl of a medium containing 5 × 10 4 cells was inoculated into a 24-well microtiter plate, and dispensed to a final concentration of 0.01%. Thereto was added 100 μl of the dispersion of the fluorescent dye-labeled polyacrylic acid-bonded titanium dioxide composite fine particles and the dispersion of the fluorescent dye-labeled photocatalytic titanium dioxide composite fine particles to a final concentration of 0.01%, respectively, and a 24-
（実施例６）
光触媒性二酸化チタン複合微粒子のｐＨ安定性の評価
５０ｍＭの異なるｐＨを持つ緩衝液（ｐＨ３＝グリシン塩酸緩衝液、ｐＨ４および５＝酢酸緩衝液、ｐＨ６＝２−モルフォリノエタンスルホン酸緩衝液）、ｐＨ７および８＝２−［４−（２−ヒドロキシエチル）−１−ピペラジニル］エタンスルホン酸緩衝液、ｐＨ９＝ホウ酸緩衝液、ｐＨ１０＝グリシン水酸化ナトリウム緩衝液）を作成し、終濃度０．０２５（ｗ／ｖ）％になるように実施例２で得られたストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子を含む分散液を添加し、1時間室温にて静置した。その後、ゼータサイザーナノＺＳにて実施例１と同様に平均分散粒径の測定を行った。結果を図５に示す。ｐＨが３から１０の間で粒径の変化は認められるものの７０から８５ｎｍ程度であり、本発明の光触媒性二酸化チタン複合微粒子を含む分散液における光触媒性二酸化チタン複合微粒子は安定した分散性を示した。
(Example 6)
Evaluation of pH stability of photocatalytic titanium dioxide composite microparticles Buffers with different pH of 50 mM (
（実施例７）
光触媒性二酸化チタン複合微粒子の塩強度安定性の評価
０．０５〜５Ｍの異なる塩化ナトリウムを含む１０ｍＭリン酸緩衝液に実施例２で得られたストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子を終濃度０．０２５％になるように添加し、１時間室温にて静置した。その後、ゼータサイザーナノＺＳにて実施例１と同様に平均分散粒径の測定を行った。結果を図６に示す。
系中の塩濃度が０．０５から１Ｍの間はほとんど平均分散粒径の変化は認められず、本発明の光触媒性二酸化チタン複合微粒子を含む分散液における光触媒性二酸化チタン複合微粒子安定した分散性を示すことが明らかになった。
(Example 7)
Evaluation of salt strength stability of photocatalytic titanium dioxide composite fine particles The photocatalytic titanium dioxide composite fine particles obtained by immobilizing the streptavidin obtained in Example 2 in a 10 mM phosphate buffer containing 0.05 to 5 M of different sodium chloride were used. The final concentration was 0.025%, and the mixture was allowed to stand at room temperature for 1 hour. Thereafter, the average dispersed particle size was measured in the same manner as in Example 1 using Zetasizer Nano ZS. The results are shown in FIG.
When the salt concentration in the system is 0.05 to 1M, almost no change in the average dispersed particle diameter is observed, and the photocatalytic titanium dioxide composite fine particles in the dispersion containing the photocatalytic titanium dioxide composite fine particles of the present invention have stable dispersibility. It became clear to show.
（実施例８）
ポリエチレンイミン結合二酸化チタン微粒子へのレクチンの固定化
実施例１で得られたポリエチレンイミン結合二酸化チタン微粒子の分散液を３０ｍＭ酢酸緩衝液（ｐＨ５．５）に懸濁し、１（ｗ／ｖ）％になるようにした。この溶液１０ｍｌに、５００ｍＭのＥＤＣ水溶液２５０μｌと１ｍｌの１ｍｇ／ｍｌのＤＢＡ（Ｄｏｌｉｃｈｏｓ Ｂｉｆｌｏｒｕｓ Ａｇｇｌｕｔｉｎｉｎ）−ＦＩＴＣ（ＶＥＣ社製：ＦＩＴＣのＤＢＡに対するモル結合比２．５)を加え、室温にて２時間攪拌を行った。反応後、２０ｍｌのイソプロパノールを加え、室温で３０分静置後、４０００ｇにて２０分間遠心分離を行った。沈殿を７０％エタノールで洗浄し、ＰＢＳ緩衝液に懸濁しＤＢＡ−ＦＩＴＣ固定化ポリエチレンイミン結合二酸化チタン微粒子の分散液を作成した。この複合体微粒子の平均分散粒子径は６８．３ｎｍであった。フルオレセイン（和光純薬工業社製）をＰＢＳ緩衝液で希釈し、励起波長５９５ｎｍ蛍光波長６２５ｎｍにて蛍光光度計で測定し、検量線を作成した。分散液の蛍光強度から６００ｎｇ／ｍｌのＦＩＴＣが結合していることが明らかになった。さらに本分散液を４００℃に加熱し、酸化チタン含量を測定したところ、１（ｗ／ｖ）％の濃度であった。ＤＢＡ：ＦＩＴＣの結合比が１：２．５であることから２．５×１０−７（ＤＢＡ−ＦＩＴＣ）ｍｏｌ／ＴｉＯ２（ｇ）であることが判明した。
(Example 8)
Immobilization of lectin on polyethyleneimine-bound titanium dioxide fine particles The dispersion of polyethyleneimine-bound titanium dioxide fine particles obtained in Example 1 was suspended in 30 mM acetate buffer (pH 5.5) to 1 (w / v)%. It was made to become. To 10 ml of this solution, 250 μl of a 500 mM EDC aqueous solution and 1 ml of 1 mg / ml DBA (Dolichos Biflorus Agglutinin) -FITC (VEC: molar ratio of FITC to DBA 2.5) were stirred for 2 hours at room temperature. Went. After the reaction, 20 ml of isopropanol was added and left at room temperature for 30 minutes, followed by centrifugation at 4000 g for 20 minutes. The precipitate was washed with 70% ethanol and suspended in PBS buffer to prepare a dispersion of DBA-FITC-immobilized polyethyleneimine-bound titanium dioxide fine particles. The average dispersed particle size of the composite fine particles was 68.3 nm. Fluorescein (manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with a PBS buffer and measured with a fluorometer at an excitation wavelength of 595 nm and a fluorescence wavelength of 625 nm to prepare a calibration curve. The fluorescence intensity of the dispersion revealed that 600 ng / ml FITC was bound. Further, this dispersion was heated to 400 ° C., and the titanium oxide content was measured. As a result, the concentration was 1 (w / v)%. Since the DBA: FITC bond ratio was 1: 2.5, it was found to be 2.5 × 10 −7 (DBA-FITC) mol / TiO 2 (g).
（実施例９）
光触媒性二酸化チタン複合微粒子を含む分散液の均一性（透明度）の評価
０．１Ｍの塩化ナトリウムを含む１０ｍＭリン酸緩衝液を用いて、実施例２で得られたストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子を含む分散液を終濃度０．１％になるように調整し、１時間室温にて静置した。また、二酸化チタン微粒子としてＰ２５（日本アエロジル）を０．１Ｍの塩化ナトリウムを含む１０ｍＭリン酸緩衝液を用いて、同様に終濃度０．１％になるように調整し、１時間室温にて静置した。その後、シャーレに５ｍｌ移し上方から撮影し、確認した。その結果を図７に示す。Ｐ２５水溶液に対してストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子を含む分散液は明らかに透明度が高く、均一に分散していることが確認された。また、分光光度計（ＵＶ−１６００、島津製作所）を用いて波長６６０ｎｍにおける吸光度の測定を行った結果、Ｐ２５水溶液は吸光度が１を大きく上回り測定不能であったのに対して、ストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子を含む分散液は吸光度が０．０４４であり、また沈殿の形成は起きていなかった。更に、これらの溶液を室温暗所にて２週間静置した後に、同様に波長６６０ｎｍにおける吸光度の測定を行った結果、Ｐ２５水溶液は吸光度が１を大きく上回り測定不能であったのに対して、ストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子を含む分散液は吸光度が０．０５１であった。このことから、水溶液中においてストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子を含む分散液が透明度の高い、均一な分散性を示し、かつ安定していることが明らかになった。
Example 9
Evaluation of Uniformity (Transparency) of Dispersion Containing Photocatalytic Titanium Dioxide Composite Fine Particles Photocatalytic property obtained by immobilizing streptavidin obtained in Example 2 using 10 mM phosphate buffer containing 0.1 M sodium chloride The dispersion containing titanium dioxide composite fine particles was adjusted to a final concentration of 0.1% and allowed to stand at room temperature for 1 hour. Further, P25 (Nippon Aerosil) as titanium dioxide fine particles was similarly adjusted to a final concentration of 0.1% using a 10 mM phosphate buffer containing 0.1 M sodium chloride, and allowed to stand at room temperature for 1 hour. I put it. Thereafter, 5 ml was transferred to a petri dish and photographed from above to confirm. The result is shown in FIG. It was confirmed that the dispersion containing the photocatalytic titanium dioxide composite fine particles in which streptavidin was immobilized on the P25 aqueous solution was clearly highly transparent and uniformly dispersed. In addition, as a result of measuring the absorbance at a wavelength of 660 nm using a spectrophotometer (UV-1600, Shimadzu Corporation), the absorbance of P25 aqueous solution was much higher than 1, whereas it was not possible to measure streptavidin. The dispersion containing the converted photocatalytic titanium dioxide composite fine particles had an absorbance of 0.044, and no precipitate was formed. Furthermore, after these solutions were allowed to stand at room temperature in a dark place for 2 weeks, the absorbance at a wavelength of 660 nm was measured in the same manner. As a result, the absorbance of the P25 aqueous solution greatly exceeded 1 and was not measurable. The dispersion containing the photocatalytic titanium dioxide composite fine particles on which streptavidin was immobilized had an absorbance of 0.051. From this, it became clear that the dispersion containing the photocatalytic titanium dioxide composite fine particles in which streptavidin is immobilized in an aqueous solution has high transparency, uniform dispersibility, and is stable.
（実施例１０）
光触媒性二酸化チタン複合微粒子を含む分散液の細胞毒性評価
実施例２で得られたストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子を含む分散液を、固形分が１．０％になるように１０％血清を含むＲＰＭＩ１６４０培地（ＧＩＢＣＯ社製）で調整した。培養ガン細胞（Ｊｕｒｋａｔ）を、１０％血清を含むＲＰＭＩ１６４０培地（ＧＩＢＣＯ社製）で３７℃、５％二酸化炭素雰囲気下で培養し、５．０×１０４ 細胞数／ｍｌとなるように調製した。これを再度２０時間同条件で培養した。この細胞培養液に、上記ストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子を含む分散液を終濃度で０．１％、０．０１％、０．００１％、０．０００１％になるように９６穴プレート上で調整し、２００μｌの試験用細胞培養液とした。この試験用細胞培養液を３７℃、５％二酸化炭素雰囲気下で２０時間培養した後、それぞれ１００μｌを用いてＣｅｌｌｔｉｔｅｒ−Ｇｌｏ Ｌｕｍｉｎｅｓｃｅｎｔ Ｃｅｌｌ Ｖｉａｂｉｌｉｔｙ Ａｓｓａｙ（Ｐｒｏｍｅｇａ社製）により生細胞由来の発光反応を行い、イメージアナライザＬＡＳ−３０００ＵＶｍｉｎｉ（富士フィルム社製）を用いてその発光量測定を行うことで細胞毒性の評価を行った。その結果を図８に示す。何も添加していないコントロールの培養細胞における発光量に比べ、どの分散液濃度においても同等の発光量を確認したことから、この濃度域のストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子を含む分散液は細胞毒性が認められないことが明らかになった。
(Example 10)
Cytotoxicity Evaluation of Dispersion Containing Photocatalytic Titanium Dioxide Composite Fine Particles The dispersion containing the photocatalytic titanium dioxide composite fine particles to which streptavidin was immobilized obtained in Example 2 was adjusted so that the solid content was 1.0%. It was prepared with RPMI 1640 medium (GIBCO) containing 10% serum. Cultured cancer cells (Jurkat) were cultured in RPMI 1640 medium (GIBCO) containing 10% serum at 37 ° C. in a 5% carbon dioxide atmosphere to prepare 5.0 × 10 4 cells / ml. . This was again cultured under the same conditions for 20 hours. In this cell culture solution, a dispersion containing the photocatalytic titanium dioxide composite fine particles to which the streptavidin is immobilized is adjusted to a final concentration of 0.1%, 0.01%, 0.001%, and 0.0001%. Prepared on a 96-well plate to give 200 μl of cell culture for testing. After culturing this test cell culture solution at 37 ° C. in a 5% carbon dioxide atmosphere for 20 hours, 100 μl each was used to perform a luminescent reaction derived from a living cell by Celltiter-Glo Luminescent Cell Viability Assay (manufactured by Promega), Cytotoxicity was evaluated by measuring the amount of luminescence using an image analyzer LAS-3000UVmini (manufactured by Fuji Film). The result is shown in FIG. Compared to the amount of luminescence in the control cultured cells to which nothing was added, the amount of luminescence was confirmed at any concentration of the dispersion, so it contains photocatalytic titanium dioxide composite fine particles with immobilized streptavidin in this concentration range. It became clear that the dispersion was not cytotoxic.
（実施例１１）
光触媒性二酸化チタン複合微粒子を含む分散液の細胞殺傷性の評価
メラノーマ細胞株Ｔ−２４を１０％血清を含むＦ１２培地（ギブコ社製）で１００％コンフルエントにになるまで培養し、フラスコを１００ｍＭリン酸緩衝食塩水（ｐＨ７．４）で２回洗浄し、１００ｍＭトリプシン−エチレンジアミン三酢酸溶液を１ｍｌ添加し、１０分静置後、フラスコ壁面より剥離した細胞を回収し、９ｍｌの１０％血清を含むＦ１２培地で希釈した。細胞数を血球計算盤により計測し、５×１０４個の細胞を含む培地５００μｌをそれぞれ２４穴マイクロタイタープレートに接種した。そこに、実施例２で得られたストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子を含む分散液を１００ｍＭリン酸緩衝食塩水（ｐＨ７．４）で調整し、最終濃度０％および０．０１％となるよう１００μｌ加え、ブラックライト（東芝社製）により波長３４０ｎｍの紫外光を２．５ｍＷ／ｃｍ２で０分間および６０分間照射し、２４時間ＣＯ２インキュベータ内で培養した。Ｃｅｌｌ ｃｏｕｎｔｉｎｇ ｋｉｔ−８（同人化学社製）を試薬のマニュアルに従い調整して加え、９６穴プレート上にて吸光度計Ｂｅｎｃｈｍａｒｋ（Ｂｉｏ−Ｒａｄ社製）を用い、波長４５０ｎｍの吸光度測定を行った。その結果を表１に示す。
(Example 11)
Evaluation of cell killing property of dispersion containing photocatalytic titanium dioxide composite fine particles Melanoma cell line T-24 was cultured in F12 medium (Gibco) containing 10% serum until 100% confluent, and the flask was treated with 100 mM phosphorus. Wash twice with acid-buffered saline (pH 7.4), add 1 ml of 100 mM trypsin-ethylenediamine triacetic acid solution, leave it for 10 minutes, collect the cells detached from the flask wall, and contain 9 ml of 10% serum Diluted with F12 medium. The number of cells was counted with a hemocytometer, and 500 μl of a medium containing 5 × 10 4 cells was inoculated on each 24-well microtiter plate. Thereto, a dispersion containing photocatalytic titanium dioxide composite fine particles having immobilized streptavidin obtained in Example 2 was adjusted with 100 mM phosphate buffered saline (pH 7.4) to obtain final concentrations of 0% and 0.01%. 100 μl was added, and ultraviolet light with a wavelength of 340 nm was irradiated at 2.5 mW /
バックグラウンドの値を差し引いた紫外線照射０分間、ストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子濃度０％における生細胞由来の吸光度を１として相対生存率を示した。この結果から、ストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子が０．０１％存在下で紫外線照射６０分間の実験条件における場合のみ、相対生存率が減少しており、これによりストレプトアビジンを固定化した光触媒性二酸化チタン複合微粒子を含む分散液は細胞殺傷性が高いことが確認された。
The relative survival rate was shown with the absorbance derived from living cells at 1% of the photocatalytic titanium dioxide composite fine particle concentration with 0% of streptavidin immobilized for 0 minutes after UV irradiation after subtracting the background value. From this result, the relative survival rate decreased only when the photocatalytic titanium dioxide composite microparticles with immobilized streptavidin were present in an experimental condition of 0.01% UV irradiation for 60 minutes, thereby fixing the streptavidin. It was confirmed that the dispersion containing the converted photocatalytic titanium dioxide composite fine particles has high cell killing ability.
本発明は、癌細胞、内分泌撹乱物質などに対する分子認識能を有するタンパク質、抗体、DNAなどの生体分子を、水溶性高分子で修飾したアナターゼ型二酸化チタンに固定することにより、これらに対する分子認識能を有し、かつ紫外線の照射などの光触媒作用によりこれら物質の分解反応を示す光触媒性二酸化チタン複合微粒子を含む分散液を提供する。本発明の光触媒性二酸化チタン複合微粒子を含む分散液における光触媒性二酸化チタン複合微粒子は水、または水溶液中で目的とする物質を特異的に認識捕捉し、紫外線照射などにより目的物質を強力に分解する能力を有する。特に水系で使用できること、目的物質を正確に捕捉できること、強力な光触媒能を有することは、例えば水系の内分泌攪乱物質の分解処理や癌細胞の破壊などの医療への応用に極めて有用である。 The present invention relates to the ability of molecular recognition for cancer cells, endocrine disrupting substances, etc. by immobilizing biomolecules such as proteins, antibodies, DNA and the like on anatase-type titanium dioxide modified with water-soluble polymers. And a dispersion containing photocatalytic titanium dioxide composite fine particles that exhibit a decomposition reaction of these substances by photocatalytic action such as ultraviolet irradiation. The photocatalytic titanium dioxide composite fine particle in the dispersion containing the photocatalytic titanium dioxide composite fine particle of the present invention specifically recognizes and captures the target substance in water or an aqueous solution, and strongly decomposes the target substance by ultraviolet irradiation or the like. Have the ability. In particular, being able to be used in an aqueous system, being able to accurately capture a target substance, and having a strong photocatalytic ability are extremely useful for medical applications such as decomposition treatment of an aqueous endocrine disrupting substance and destruction of cancer cells.
Claims (20)
The dispersion liquid according to any one of claims 1 to 19, wherein the dispersion liquid contains 0.0001 to 0.1% of the photocatalytic titanium dioxide composite fine particles by weight.
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| PCT/JP2006/306179 WO2007034586A1 (en) | 2005-09-22 | 2006-03-27 | Photocatalytic titanium dioxide microparticle, dispersion liquid thereof and process for producing the same |
| US11/883,212 US20080261805A1 (en) | 2005-09-22 | 2006-03-27 | Photocatalytic Titanium Dioxide Microparticle, Dispersion Liquid Thereof and Process for Producing the Same |
| US12/943,165 US20110060269A1 (en) | 2005-09-22 | 2010-11-10 | Method for killing cells using photocatalytic titanium dioxide particles |
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