JP7061815B2 - Microaggregates and their manufacturing methods - Google Patents
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- JP7061815B2 JP7061815B2 JP2020519603A JP2020519603A JP7061815B2 JP 7061815 B2 JP7061815 B2 JP 7061815B2 JP 2020519603 A JP2020519603 A JP 2020519603A JP 2020519603 A JP2020519603 A JP 2020519603A JP 7061815 B2 JP7061815 B2 JP 7061815B2
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Description
本発明は、微小会合体およびその製造方法に係り、特に天然植物由来のタンニン酸の誘導体を用いて自己組織化された微小会合体およびその製造方法に関する。 The present invention relates to a microaggregate and a method for producing the same, and more particularly to a microaggregate self-assembled using a derivative of tannic acid derived from a natural plant and a method for producing the same.
内空部に水などの液体を有し、その外側に外殻が形成されていて、その外殻は外側が親水性で内部が疎水性の性質をもつ膜からなる構造の微小会合体がある。
そして、この構造の微小会合体を、内容物を運ぶための微小カプセルとして利用する試みが盛んになされている。例えば、この微小カプセルの内部あるいは外殻部に薬剤などを担持させ、通常状態ではその薬剤をカプセルにより保護し、界面活性剤などによるある特定の化学的刺激によりカプセルを解離させて薬剤を供給する方法が検討されている（特許文献１参照）。
ここで、この会合体（カプセル）に担持させるものは医療用の薬剤に限らない。担持させるものを酵素などとすることにより、バイオ燃料電池、バイオセンサー、エネルギー変換素子、分析法などへの応用も可能になる（特許文献２，３および非特許文献１、２参照）。It has a liquid such as water in the inner space, and an outer shell is formed on the outer side. The outer shell has a microaggregate structure consisting of a membrane having hydrophilic properties on the outside and hydrophobic properties on the inside. ..
Attempts have been made to utilize the micro-aggregates of this structure as microcapsules for carrying the contents. For example, a drug or the like is supported inside or in the outer shell of the microcapsule, the drug is protected by the capsule under normal conditions, and the capsule is dissociated by a specific chemical stimulus such as a surfactant to supply the drug. A method has been studied (see Patent Document 1).
Here, what is carried on this aggregate (capsule) is not limited to a medical drug. By using an enzyme or the like as a carrier, it can be applied to biofuel cells, biosensors, energy conversion elements, analytical methods, etc. (see
このように、医療分野を始め様々な分野で応用可能な微小会合体であるが、その製造方法としては、自己組織化メカニズムを用いた方法が多く試みられている（非特許文献３，４参照）。自己組織化を利用することにより、効率よく微小構造体を製造することができる。
As described above, although it is a microaggregate that can be applied in various fields including the medical field, many attempts have been made to use a self-organizing mechanism as a manufacturing method thereof (see Non-Patent
上述の微小会合体として検討が行われてきた代表例としては、リポソームを挙げることができる（特許文献１参照）。リポソームは、狭義には、外殻部がリン脂質で構成された会合体である。
しかしながら、外殻部を構成する材料がリン脂質に限定されると、適用範囲も限られてしまう。Liposomes can be mentioned as a typical example that has been studied as the above-mentioned microaggregates (see Patent Document 1). Liposomes are, in a narrow sense, aggregates whose outer shell is composed of phospholipids.
However, if the material constituting the outer shell portion is limited to phospholipids, the scope of application is also limited.
そこで、発明者は、天然植物由来の材料で構成される外殻部をもつ会合体の研究を行った。外殻部を天然植物由来の材料とすることにより、より人体や環境に馴染み、害が少ないものになることが期待される。 Therefore, the inventor studied an aggregate having an outer shell composed of a material derived from a natural plant. By using the outer shell as a material derived from natural plants, it is expected that it will be more familiar to the human body and the environment and will be less harmful.
本発明が解決しようとする課題は、薬剤などのキャリアカプセルとしての機能を有し、化学的刺激で容易に解離し、かつその外殻部が天然植物由来の材料で構成される微小会合体およびその製造方法を提供することである。 The problem to be solved by the present invention is a microaggregate having a function as a carrier capsule for a drug or the like, easily dissociated by a chemical stimulus, and its outer shell made of a material derived from a natural plant. It is to provide the manufacturing method.
本発明の構成を下記に示す。
（構成１）
最外部が殻構造を形成している微小会合体であって、
前記殻構造は、タンニン酸の少なくとも一部の水酸基における水素原子が直鎖の炭化水素基で置換されたタンニン酸誘導体の２分子膜構造体からなり、
前記直鎖の炭化水素基の炭素数が６以上１６以下である、微小会合体。
（構成２）
前記タンニン酸誘導体の前記直鎖の炭化水素基による置換数が５以上２５以下である、構成１記載の微小会合体。
（構成３）
前記タンニン酸誘導体の前記直鎖の炭化水素基による置換数が１０である、構成２記載の微小会合体。
（構成４）
前記殻構造の厚さが３ｎｍ以上６ｎｍ以下である、構成１から３の何れか１に記載の微小会合体。
（構成５）
外形の形状が球形である、構成１から４の何れか１に記載の微小会合体。
（構成６）
前記外形の直径が１００ｎｍ以上２００ｎｍ以下である、構成５に記載の微小会合体。
（構成７）
前記殻構造で囲まれた内空部が液体で満たされている、構成１から６の何れか１に記載の微小会合体。
（構成８）
前記殻構造で囲まれた内空部に空間が形成されている、構成１から６の何れか１に記載の微小会合体。
（構成９）
前記殻構造で囲まれた内空部に薬剤が担持された、構成１から８の何れか１に記載の微小会合体。
（構成１０）
前記殻構造を構成する膜に薬剤が担持された、構成１から８の何れか１に記載の微小会合体。
（構成１１）
前記薬剤は、食物発酵生成物、レシチン、イソフラボン、カテキン、ガロカテキン、カテキン３－ガレート、ガロカテキン３－ガレート、エピカテキン、エピガロカテキン、エピカテキン３－ガレート、シリマリン、クルクミノイド、ギンゲロール、セラミド；イソプレン、プレノール、イソ吉草酸、ゲラニルピロホスフェート、オイカリプトール、リモネン、ピネン、ファルネシルピロホスフェート、アルテミシニン、ビサボロール、ゲラニルゲラニルピロホスフェート、レチノール、レチナール、フィトール、タキソール、フォルスコリン、アフィジコリン、スクアレン、ラノステロール、テルペン、植物性油またはアマランス種子もしくは米由来の油、レチノイド、ケイ皮酸、リグニン、ポリフェノール、ビタミン、カフェニン、テオブロミン、メチル－、ジメチル－およびパラ－ルキサンチン、キサンチンアルカロイド、ペニシリン、真菌代謝物、セファロスポリン、カルダペネム、スルホンアミド、キノロン、オキサゾジノン、マクロライド、抗ウイルス薬、心血管薬、代謝薬、抗真菌薬、および抗寄生虫薬、ならびにスタチンの群から選ばれる少なくとも１以上である、構成９または１０に記載の微小会合体。
（構成１２）
タンニン酸の少なくとも一部の水酸基における水素原子が直鎖の炭化水素基で置換されたタンニン酸誘導体を準備するタンニン酸誘導体準備工程と、
前記タンニン酸誘導体を良溶媒に溶解させる溶解工程と、
前記タンニン酸誘導体が溶解した良溶媒に貧溶媒を添加する貧溶媒添加工程とを有する、
構成１から１１の何れか１に記載の微小会合体を製造する微小会合体製造方法。
（構成１３）
前記良溶媒はテトラヒドロフラン、アセトン、イソプロピルアルコールの群から選ばれる少なくとも１であり、前記貧溶媒は純水である、構成１２に記載の微小会合体製造方法。
（構成１４）
前記タンニン酸誘導体の前記良溶媒に対する比率は０．０１ｍｇ／ｍＬ以上１．２０ｍｇ／ｍＬ以下であり、前記貧溶媒の添加速度は、２ｍＬ／ｍｉｎ以上６００ｍＬ／ｍｉｎ以下である、構成１２または１３に記載の微小会合体製造方法。
The configuration of the present invention is shown below.
(Structure 1)
The outermost is a microaggregate forming a shell structure,
The shell structure consists of a bilayer film structure of a tannic acid derivative in which hydrogen atoms at at least a part of the hydroxyl groups of tannic acid are replaced with linear hydrocarbon groups.
A microaggregate having 6 or more and 16 or less carbon atoms in the linear hydrocarbon group .
(Structure 2)
The microaggregate according to
(Structure 3)
The microaggregate according to
(Structure 4)
The microaggregate according to any one of
(Structure 5)
The microaggregate according to any one of
(Structure 6)
The microaggregate according to
(Structure 7)
The microaggregate according to any one of
(Structure 8)
The microaggregate according to any one of
(Structure 9)
The microaggregate according to any one of
(Structure 10)
The microaggregate according to any one of
(Structure 11)
The agents include food fermentation products, lecithin, isoflavone, catechin, galocatechin, catechin 3-gallate, galocatechin 3-gallate, epicatechin, epigalocatechin, epicatechin 3-gallate, silimarin, curcuminoid, gingerol, ceramide; isoprene, Plenol, isovaleric acid, geranylpyrophosphate, eucalyptoll, limonen, pinen, farnesylpyrophosphate, artemicinin, bisabolol, geranylgeranylpyrophosphate, retinol, retinal, phytol, taxol, forskolin, aphidicholine, squalene, lanosterol, terpene Or oils from Amaranth seeds or rice, retinoids, silicic acid, lignin, polyphenols, vitamins, caffeine, theobromine, methyl-, dimethyl- and para-lucanthin, xanthin alkaloids, penicillins, fungal metabolites, cephalosporins, cardapenems , Sulphonamide, quinolone, oxazodinone, macrolides, antiviral agents, cardiovascular agents, metabolites, antifungal agents, and antiparasitic agents, and at least one selected from the group of statins, in
(Structure 12)
A tannic acid derivative preparation step for preparing a tannic acid derivative in which a hydrogen atom in at least a part of the hydroxyl groups of tannic acid is replaced with a linear hydrocarbon group, and a step of preparing the tannic acid derivative.
A dissolution step of dissolving the tannic acid derivative in a good solvent, and
It comprises a poor solvent addition step of adding a poor solvent to a good solvent in which the tannic acid derivative is dissolved.
The method for producing a microaggregate according to any one of
(Structure 13)
The method for producing a microaggregate according to
(Structure 14)
The ratio of the tannic acid derivative to the good solvent is 0.01 mg / mL or more and 1.20 mg / mL or less, and the addition rate of the poor solvent is 2 mL / min or more and 600 mL / min or less. The method for producing a microaggregate according to .
本発明により、人体、環境に馴染みやすい天然由来の植物原料を基に、薬剤等をカプセル状化して担持可能な自己組織化された微小会合体、およびその製造方法を提供することが可能になる。また、このカプセル状微小会合体は、界面活性剤による化学的刺激により容易に解離させることができるので、薬剤等のキャリアとして優れた効果を発揮する。 INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to provide a self-organized microaggregate capable of encapsulating and supporting a drug or the like based on a naturally-derived plant material that is easily compatible with the human body and the environment, and a method for producing the same. .. Further, since the capsule-shaped microaggregates can be easily dissociated by chemical stimulation with a surfactant, they exert an excellent effect as a carrier of a drug or the like.
１．本発明の会合体のコンセプト
本発明の微小会合体は、図１に示すように、天然ポリフェノールを用いた両親媒性デンドリマーを外殻部に有し、内空部が水あるいは親水性の極性溶媒で満たされた球形の会合体である。そして、この両親媒性デンドリマーは、図２に示すように、タンニン酸からなる親水性コアの部分と直鎖の炭化水素基からなる疎水性リムの部分からなる。1. 1. Concept of the aggregate of the present invention As shown in FIG. 1, the micro-aggregate of the present invention has an amphipathic dendrimer using a natural polyphenol in the outer shell, and the inner space is water or a hydrophilic polar solvent. It is a spherical assembly filled with. And, as shown in FIG. 2, this amphipathic dendrimer consists of a hydrophilic core portion made of tannic acid and a hydrophobic rim portion made of a linear hydrocarbon group.
この微小会合体は、薬剤や試薬などを外殻部あるいは内空部に担持することができて、薬剤や試薬などを保護するいわゆるカプセルの役割を担うことができる。
また、この微小会合体は、界面活性剤などの化学的刺激を受けると外殻部が解離し、微小会合体は解体される。この解離ないし解体に伴い、外殻部あるいは内空部に担持されていた薬剤や試薬などを放出することができるため、薬剤や試薬等のデリバリーに適する。This micro-aggregate can carry a drug, a reagent, or the like on an outer shell portion or an inner space portion, and can play a role of a so-called capsule that protects the drug, the reagent, or the like.
Further, when the micro-aggregate is subjected to a chemical stimulus such as a surfactant, the outer shell portion is dissociated and the micro-aggregate is disassembled. With this dissociation or disassembly, the drug or reagent supported on the outer shell or the inner space can be released, which is suitable for delivery of the drug or reagent.
すなわち、この微小会合体は、通常の状態ではカプセルとして薬剤や試薬などを保護し、狭い場所に対しても容易に輸送される。そして、化学的刺激が与えられると解体し、担持されていた薬剤や試薬等が、その場に制御性をもって供給される。
この微小会合体は、このようなデリバリーシステムを与えるものとなっている。なお、微小会合体に担持する薬剤や試薬は、親水性のものでも疎水性のものでもよく、両対応となっている。That is, the microaggregates protect drugs, reagents, etc. as capsules under normal conditions, and are easily transported even in a narrow space. Then, when a chemical stimulus is given, it is disassembled, and the carried drug, reagent, or the like is supplied to the place with controllability.
This microaggregate provides such a delivery system. The drug or reagent carried on the microaggregate may be hydrophilic or hydrophobic, and both are compatible.
外殻部を構成するタンニン酸誘導体の膜は、図３に示すように、その膜の内側でリム部の疎水性アルコキシ基が結びついた２層構造を有する。このとき、外側に向かって芳香族水酸基が露出するため、この微小会合体に薬剤を担持させない場合でも、抗菌性を有する。さらに、この微小会合体に、薬剤として滅菌剤を担持させた場合は、両者の効果が加わった滅菌効果が発揮される。 As shown in FIG. 3, the film of the tannic acid derivative constituting the outer shell portion has a two-layer structure in which the hydrophobic alkoxy group of the rim portion is bonded inside the film. At this time, since the aromatic hydroxyl group is exposed toward the outside, it has antibacterial properties even when the drug is not supported on the microaggregates. Further, when a sterilizing agent is carried as a drug on the microaggregates, the sterilizing effect is exhibited by adding the effects of both.
この微小会合体は、溶媒による自己組織化、自己会合特性を有し、この自己組織化によりサイズが制御される特徴を有する。 This micro-aggregate has the characteristics of self-assembly and self-association by a solvent, and the size is controlled by this self-assembly.
２．会合体の構造
本発明の微小会合体は、図３に示すように、親水性コアおよび疎水性アルコキシ基（直鎖の炭化水素基）を有する外殻部と、その内側の内空部とを有する。ここで、親水性コアはタンニン酸で構成される。
その外殻部の外側および内側の表面にはそれぞれ、タンニン酸の芳香族水酸基が露出するため、微小会合体は親水性となる。また内空部と接する表面も親水性となる。一方、外殻を構成する膜の中央部は疎水性となる。
なお、内空部には水や水溶液が入る。2. 2. Structure of the aggregate As shown in FIG. 3, the microaggregate of the present invention has an outer shell portion having a hydrophilic core and a hydrophobic alkoxy group (a linear hydrocarbon group), and an inner space portion inside the outer shell portion. Have. Here, the hydrophilic core is composed of tannic acid.
Since the aromatic hydroxyl groups of tannic acid are exposed on the outer and inner surfaces of the outer shell, the microaggregates become hydrophilic. The surface in contact with the inner space is also hydrophilic. On the other hand, the central portion of the membrane constituting the outer shell becomes hydrophobic.
Water or an aqueous solution enters the inner space.
外殻部は、タンニン酸の少なくとも一部の水酸基における水素原子が直鎖の炭化水素基で置換されたタンニン酸誘導体の２分子膜を有する構造、好ましくはタンニン酸誘導体の２分子膜からなる構造になっている。 The outer shell portion has a structure having a bilayer film of a tannin acid derivative in which a hydrogen atom at at least a part of the hydroxyl groups of tannin acid is substituted with a linear hydrocarbon group, preferably a structure composed of a bilayer film of a tannin acid derivative. It has become.
タンニン酸誘導体は、タンニンを原材料にして製造することができる。
タンニンは、加水分解で多価フェノールを生じる植物成分の総称であり、没食子酸やエラグ酸がグルコースなどにエステル結合し、酸や酵素で加水分解されやすい加水分解型タンニンと、フラバノール骨格を持つ化合物が重合した縮合型タンニンとに大別される。いずれのタイプのタンニンであっても、また、それらの混合物であっても、本発明におけるタンニン酸誘導体を作製すること（誘導体化）は可能であり、本発明の効果が奏されるものと考えられる。好ましくは加水分解型タンニンであり、例えば下記式（Ａ１）で表されるタンニン酸（以後ＴＡとも称す）を主成分とするものが誘導体化される。誘導化されたタンニン酸誘導体の一例を下記式（Ａ２）に示す。これは、式（Ａ１）のタンニン酸の１０個の水酸基が、炭素数１６の直鎖の炭化水素基で置換されたタンニン酸誘導体である。The tannic acid derivative can be produced using tannin as a raw material.
Tannin is a general term for plant components that produce polyhydric phenol by hydrolysis. Hydrolysable tannin in which gallic acid and ellagic acid are ester-bonded to glucose and are easily hydrolyzed by acids and enzymes, and compounds having a flavanol skeleton. Is roughly classified into polymerized condensed tannins. It is possible to produce (derivatize) the tannic acid derivative of the present invention regardless of the type of tannin or a mixture thereof, and it is considered that the effect of the present invention is exhibited. Will be. Hydrolyzable tannin is preferable, and for example, one containing tannic acid (hereinafter, also referred to as TA) represented by the following formula (A1) as a main component is derivatized. An example of the derivatized tannic acid derivative is shown in the following formula (A2). This is a tannic acid derivative in which the 10 hydroxyl groups of the tannic acid of the formula (A1) are replaced with a linear hydrocarbon group having 16 carbon atoms.
このタンニン酸誘導体における直鎖の炭化水素基による置換数は、５以上２５以下が好ましく、６以上１６以下がより好ましく、１０が最も好ましい。置換数が５未満の場合は、後述する自己組織化の条件によっては十分な外殻を形成できないことがあり、置換数が２５を超える場合は沈殿物を生じやすい。
また、このタンニン酸誘導体のリム部を構成する直鎖の炭化水素基の炭素の数は６以上１６以下が好ましい。直鎖の炭化水素基の炭素の数がこの範囲にあると、微小会合体を形成するために適切な会合力が得られるという効果がある。
このタンニン酸誘導体のリム部を構成する直鎖の炭化水素基は、炭素の数は６以上１６以下であれば、１種類である必要はない。例えば、炭素数６の直鎖の炭化水素基と炭素数１６の直鎖の炭化水素基とが混在してタンニン酸の置換がなされていてもよい。但し、炭素数の揃っている直鎖の炭素水素基を用いる方が、タンニン酸誘導体を製造しやすく、製造されるタンニン酸誘導体の品質を安定させやすい。一方で、外殻部に薬剤を担持させる場合は、炭素数の異なる複数の直鎖の炭化水素基を混在させたタンニン酸誘導体の方が、薬剤を担持させやすいという効果がある。The number of substitutions with the linear hydrocarbon group in this tannic acid derivative is preferably 5 or more and 25 or less, more preferably 6 or more and 16 or less, and most preferably 10. When the number of substitutions is less than 5, a sufficient outer shell may not be formed depending on the conditions of self-organization described later, and when the number of substitutions exceeds 25, a precipitate is likely to be formed.
Further, the number of carbon atoms of the linear hydrocarbon group constituting the rim portion of this tannic acid derivative is preferably 6 or more and 16 or less. When the number of carbons in the linear hydrocarbon group is in this range, there is an effect that an appropriate associative force can be obtained to form a microaggregate.
The linear hydrocarbon group constituting the rim portion of the tannic acid derivative does not need to be one kind as long as the number of carbons is 6 or more and 16 or less. For example, a linear hydrocarbon group having 6 carbon atoms and a linear hydrocarbon group having 16 carbon atoms may be mixed and substituted with tannic acid. However, it is easier to produce a tannic acid derivative and to stabilize the quality of the produced tannic acid derivative by using a linear carbon hydrogen group having the same number of carbon atoms. On the other hand, when a drug is supported on the outer shell, a tannic acid derivative in which a plurality of linear hydrocarbon groups having different carbon atoms are mixed has an effect that the drug can be easily supported.
タンニン酸誘導体の分子構造とそのサイズとを、密度汎関数法（ＤＦＴ：Ｄｅｎｓｉｔｙ Ｆｕｎｃｔｉｏｎａｌ Ｔｈｅｏｒｙ）によって求めた一例を図４に示す。ここで、ＴＡ（Ｃｘ）１０は、タンニン酸（ＴＡ）の水酸基のうちの１０個が、炭素数ｘの直鎖の炭化水素基によって置換されたことを示す。したがって、図４は、各々、炭素数が６、１０および１６の炭化水素基１０個により置換されたタンニン酸誘導体の分子構造とそのサイズを計算した結果になっている。この結果から、タンニン酸誘導体ＴＡ（Ｃ６）１０、ＴＡ（Ｃ１０）１０、ＴＡ（Ｃ１６）１０の直径は、各々約３．５ｎｍ、４．３ｎｍおよび５．３ｎｍと見積もられる。
これらのタンニン酸誘導体が２分子膜構造の外殻部を形成する場合は、分子の枝の部分が折り曲げられてコアの芳香族水酸基を有する部分が外側にくる分子の形で２層膜構造を作ること、および後程述べる実施例で測定した結果からみて、外殻部の厚さは、３ｎｍ以上６ｎｍ以下、代表的には約５．２ｎｍとなる。FIG. 4 shows an example in which the molecular structure of the tannic acid derivative and its size are determined by the density functional theory (DFT) method. Here, TA (C x ) 10 indicates that 10 of the hydroxyl groups of tannic acid (TA) have been replaced by a linear hydrocarbon group having x carbon atoms. Therefore, FIG. 4 is the result of calculating the molecular structure and the size of the tannic acid derivative substituted with 10 hydrocarbon groups having 6, 10 and 16 carbon atoms, respectively. From this result, the diameters of the tannic acid derivatives TA (C 6 ) 10 , TA (C 10 ) 10 , and TA (C 16 ) 10 are estimated to be about 3.5 nm, 4.3 nm, and 5.3 nm, respectively.
When these tannin acid derivatives form the outer shell part of the bilayer membrane structure, the bilayer membrane structure is formed in the form of a molecule in which the branch portion of the molecule is bent and the portion having the aromatic hydroxyl group of the core is on the outside. From the results of making and measuring in the examples described later, the thickness of the outer shell portion is 3 nm or more and 6 nm or less, typically about 5.2 nm.
この微小会合体は自己組織化により形成される。数多くの実験を行った結果、この微小会合体が１００ｎｍ以上２００ｎｍの大きさのときに、凝集体になりにくく、また大きさのばらつきが少なくなること見出した。 This microaggregate is formed by self-organization. As a result of many experiments, it was found that when the microaggregates have a size of 100 nm or more and 200 nm, they are less likely to form aggregates and the size variation is reduced.
微小会合体の外形は、このような微細な大きさであることと、外殻部の界面張力が形状を決定する上での主要因になることから、球形である。なお、ここでの球形とは、最も大きな長径が、平均した直径の１２０％以内に収まる形状をいう。 The outer shape of the microaggregate is spherical because it has such a fine size and the interfacial tension of the outer shell is a main factor in determining the shape. The spherical shape here means a shape in which the largest major axis is within 120% of the average diameter.
本微小会合体において薬剤等が担持される場所は、図５に示すように、その薬剤が親水性であるか疎水性であるかによって異なる。薬剤が親水性である場合は、内空部に水や水溶液からなる液体とともに存在し、薬剤が疎水性である場合は、外殻部を構成する膜（外郭膜）の中に存在する。 As shown in FIG. 5, the place where the drug or the like is supported in the microaggregate differs depending on whether the drug is hydrophilic or hydrophobic. When the drug is hydrophilic, it exists in the inner space together with a liquid consisting of water or an aqueous solution, and when the drug is hydrophobic, it exists in the membrane (outer membrane) constituting the outer shell.
担持する薬剤としては、例えば、食物発酵生成物、レシチン、イソフラボン、カテキン、ガロカテキン、カテキン３－ガレート、ガロカテキン３－ガレート、エピカテキン、エピガロカテキン、エピカテキン３－ガレート、シリマリン、クルクミノイド、ギンゲロール、セラミド、イソプレン、プレノール、イソ吉草酸、ゲラニルピロホスフェート、オイカリプトール、リモネン、ピネン、ファルネシルピロホスフェート、アルテミシニン、ビサボロール、ゲラニルゲラニルピロホスフェート、レチノール、レチナール、フィトール、タキソール、フォルスコリン、アフィジコリン、スクアレン、ラノステロール、テルペン、植物性油またはアマランサス種子もしくは米由来の油、レチノイド、ケイ皮酸、リグニン、ポリフェノール、ビタミン、カフェイン、テオブロミン、メチル－、ジメチル－およびパラ－キサンチン、キサンチンアルカロイド、ペニシリン、真菌代謝物、セファロスポリン、カルダペネム、スルホンアミド、キノロン、オキサゾリジノン、マクロライド、抗ウイルス薬、心血管薬、代謝薬、抗真菌薬、および抗寄生虫薬、ならびにスタチンの群から選ばれる少なくとも１以上を挙げることができる。 Examples of the drug to be carried include food fermentation products, lecithin, isoflavone, catechin, galocatechin, catechin 3-gallate, galocatechin 3-gallate, epicatechin, epigalocatechin, epicatechin 3-gallate, silymarin, curcuminoid, gingerol, and the like. Ceramide, isoprene, plenol, isovaleric acid, geranylpyrophosphate, eucalyptoll, limonen, pinen, farnesylpyrophosphate, artemicinin, bisabolol, geranylgeranylpyrophosphate, retinol, retinal, phytol, taxol, forskolin, affidicholine, squalene, , Vegetable oil or oil from amaranthus seeds or rice, retinoids, cinnate, lignin, polyphenols, vitamins, caffeine, theobromine, methyl-, dimethyl- and para-xanthin, xanthin alkaloids, penicillins, fungal metabolites, cephalo At least one selected from the group of sporins, cardapenems, sulfonamides, quinolones, oxazolidinones, macrolides, antiviral agents, cardiovascular agents, metabolites, antifungal agents, and antiparasitic agents, and statins can be mentioned. ..
３．会合体の製造方法
最初に、タンニン酸（ＴＡ）の少なくとも一部の水酸基における水素原子が直鎖の炭化水素基で置換されたタンニン酸誘導体（ＰＡＴＡ：Ｐａｒｔｉａｌｌｙ ｎ－ａｌｋｙｌ ｓｕｂｓｔｉｔｕｔｅｄ ＴＡ）を作製、準備するタンニン酸誘導体作製工程を行う。
このタンニン酸誘導体は、アルキル化反応の一つであるウィリアムソンエーテル合成法によって得ることができる。具体的には、テトラヒドロフラン、ジメチルスホキサイド等の溶媒中で、塩基性触媒の存在下で、タンニン酸にハロゲン化アルキルを反応させて作ることができる。塩基性触媒としてはＭＨ、Ｍ２ＣＯ３、Ｍ（Ｍ：アルカリ金属）の群から選択されるいずれか１または２以上の触媒を使うことができる。例えば、Ｋ２ＣＯ３は、ＯＨ基をＯ－Ｋ＋に変換し、ハロゲン化アルキル（Ｘ-Ｒ１：Ｘ：ハロゲン、Ｒ１：アルキル基）へのＯ－基の求核反応を促進することができる。ハロゲン化アルキルとしては、例えば、ヨウ化アルキルを用いることができる。また、ハロゲン化アルキルの代わりに、スルホニル基などを脱離基として有するものも使用できる。また、上記、ウィリアムソンエーテル合成法以外のアルキル化反応を用いることもできる。さらに、Ｎ，Ｎ′－ジシクロヘキシルカルボジイミド（ＤＯＣ）等の縮合剤を用いたカルボン酸類との脱水縮合反応や、イソシアネートとの縮合反応を用いることもできる。3. 3. Method for producing aggregate First, a tannin acid derivative (PATA: Partially n-alkyl substituted TA) in which a hydrogen atom in at least a part of hydroxyl groups of tannin acid (TA) is replaced with a linear hydrocarbon group is prepared and prepared. The step of producing a tannin acid derivative is carried out.
This tannic acid derivative can be obtained by the Williamson ether synthesis method, which is one of the alkylation reactions. Specifically, it can be produced by reacting tannic acid with an alkyl halide in the presence of a basic catalyst in a solvent such as tetrahydrofuran or dimethylsucciside. As the basic catalyst, any one or more catalysts selected from the group of MH, M 2 CO 3 , and M (M: alkali metal) can be used. For example, K 2 CO 3 converts the OH group to OK + and promotes the nucleophilic reaction of the O- group to the alkyl halide (X-R 1 : X: halogen, R 1 : alkyl group). be able to. As the alkyl halide, for example, an alkyl iodide can be used. Further, instead of the alkyl halide, those having a sulfonyl group or the like as a leaving group can also be used. Further, an alkylation reaction other than the above-mentioned Williamson ether synthesis method can also be used. Further, a dehydration condensation reaction with carboxylic acids using a condensing agent such as N, N'-dicyclohexylcarbodiimide (DOC) or a condensation reaction with isocyanate can also be used.
反応は、例えば、７０℃以上１００℃以下で、約１時間程度加熱する。タンニン酸に対するハロゲン化アルキルのモル比を変えることにより、アルキル基のタンニン酸中への導入数であるｎの値を所望の値に設定できる。 The reaction is heated at, for example, 70 ° C. or higher and 100 ° C. or lower for about 1 hour. By changing the molar ratio of the alkyl halide to the tannic acid, the value of n, which is the number of alkyl groups introduced into the tannic acid, can be set to a desired value.
次に、液体中での自己組織化反応工程を行って、微小会合体を作製する。
この自己組織化反応工程は、大別すると、タンニン酸誘導体を良溶媒に溶解させる溶解工程と、タンニン酸誘導体が溶解した良溶媒に貧溶媒を添加する貧溶媒添加工程からなる。
その代表的な工程を下記に示す。Next, a self-assembling reaction step in a liquid is performed to prepare microaggregates.
This self-assembling reaction step is roughly divided into a dissolution step of dissolving the tannin acid derivative in a good solvent and a poor solvent addition step of adding a poor solvent to the good solvent in which the tannin acid derivative is dissolved.
The typical process is shown below.
溶解工程では、上記方法で作製したタンニン酸誘導体を良溶媒に溶解させる。ここで、良溶媒としてはテトラヒドロフラン（ＴＨＦ）、アセトン、イソプロピルアルコールの群から選ばれる少なくとも１を挙げることができる。タンニン酸誘導体の良溶媒に対する比率は０．０１ｍｇ／ｍＬ以上１．２０ｍｇ／ｍＬ以下が好ましい。比率がこの範囲に収まっていると、凝集が少ない微小会合体が形成されやすく、微小会合体の大きさのばらつきも少ないものとなる。また、この比率が小さいほど微小会合体の大きさは小さくなる。一方で、０．４ｍｇ／ｍＬを超えると微小会合体の大きさは飽和し、大きくなりにくい。
この溶解工程は室温（２５℃）で行うことができる。ここで、均一な溶解液を作るために、スターラーなどにより攪拌させて溶解させることが好ましい。In the dissolution step, the tannic acid derivative prepared by the above method is dissolved in a good solvent. Here, as a good solvent, at least one selected from the group of tetrahydrofuran (THF), acetone, and isopropyl alcohol can be mentioned. The ratio of the tannic acid derivative to a good solvent is preferably 0.01 mg / mL or more and 1.20 mg / mL or less. When the ratio is within this range, microaggregates with less aggregation are likely to be formed, and the size variation of the microaggregates is also small. Also, the smaller this ratio, the smaller the size of the microaggregates. On the other hand, when it exceeds 0.4 mg / mL, the size of the microaggregate is saturated and it is difficult to increase.
This melting step can be performed at room temperature (25 ° C.). Here, in order to prepare a uniform dissolution liquid, it is preferable to dissolve by stirring with a stirrer or the like.
貧溶媒添加工程では、溶解工程で作製したタンニン酸誘導体が溶解した溶解液に貧溶媒を添加する。最初に所定の量の貧溶媒を添加し、その後一定の速度で貧溶媒を添加する。ここで、貧溶媒としては、水及び炭素数１～４の低級アルコールの群から選ばれる少なくとも１を挙げることができ、中でも水が好ましく、純水がより好ましい。ここで、純水とは、電気抵抗率が０．１ＭΩ・ｃｍ以上の水をいう。
最初に添加する貧溶媒の量は、凝集の少ない微小会合体を形成する上で重要で、溶解液と貧溶媒との合計に対し、貧溶媒の量が７５重量％以上であることが好ましい。初期の貧溶媒の添加量が少ない場合は、皴がよった形状の微小会合体になりやすい。
貧溶媒の添加速度は、２ｍＬ／ｍｉｎ以上６００ｍＬ／ｍｉｎ以下が好ましい。貧溶媒の添加速度が２ｍＬ／ｍｉｎを下回ると微小会合体が沈殿しやすく、６００ｍＬ／ｍｉｎを上回ると作製される微小会合体のサイズが小さくなって、微小会合体の製造効率、収率が下がる。
なお、均一性を上げるために、タンニン酸誘導体が溶解した溶液をスターラーなどにより攪拌させた状態で、貧溶媒を添加することが好ましい。また、貧溶媒を添加する一方で、ゆっくりとＴＨＦなどの揮発性の溶媒を蒸散させることが好ましい。In the poor solvent addition step, the poor solvent is added to the solution in which the tannic acid derivative prepared in the dissolution step is dissolved. First, a predetermined amount of the poor solvent is added, and then the poor solvent is added at a constant rate. Here, as the poor solvent, at least one selected from the group of water and lower alcohols having 1 to 4 carbon atoms can be mentioned, and water is preferable, and pure water is more preferable. Here, pure water means water having an electrical resistivity of 0.1 MΩ · cm or more.
The amount of the poor solvent added first is important for forming microaggregates with less aggregation, and the amount of the poor solvent is preferably 75% by weight or more with respect to the total of the solution and the poor solvent. When the amount of the initial poor solvent added is small, the microaggregates tend to have a wrinkled shape.
The addition rate of the poor solvent is preferably 2 mL / min or more and 600 mL / min or less. When the addition rate of the poor solvent is less than 2 mL / min, the microaggregates are likely to precipitate, and when it exceeds 600 mL / min, the size of the microaggregates produced becomes smaller, and the production efficiency and yield of the microaggregates decrease. ..
In order to improve the uniformity, it is preferable to add the poor solvent in a state where the solution in which the tannic acid derivative is dissolved is stirred with a stirrer or the like. Further, it is preferable to slowly evaporate a volatile solvent such as THF while adding a poor solvent.
４．会合体の解離方法と薬剤、試薬のデリバリー
薬剤を担持した微小会合体の作製から解離による薬剤放出に至る過程を図６に示す。
微小会合体への薬剤の担持は、ＴＨＦなどの良溶媒中にタンニン酸誘導体（ＰＡＴＡ）が溶解している段階で薬剤を添加し、その後、上述の方法で貧溶媒を添加して、微小会合体を液中形成することで行う。この段階で、微小会合体はカプセル状になって、担持された薬剤を保護する。この微小会合体は水中などでも安定である。
微小会合体が水などの液体中にある状態で、界面活性剤による化学的刺激を受けると、外殻を構成している膜の分子間の結びつきが解けて解離し、微小会合体の内空部あるいは外殻部に担持されていた薬剤が液中に放出される。ここで、界面活性剤としては、例えば、ＣＴＡＢ（Ｃｅｔｙｌｔｒｉｍｅｔｈｙｌ ａｍｍｏｎｉｕｍ ｂｒｏｍｉｄｅ）、ｎ－ブチルアミン、ｎ－ヘキシルアミン、ジイソプロピルアミン、トリエチルアミン、アニリン、テトラ－ｎ－ブチルアンモニウムブロマイドなどを挙げることができる。4. Dissociation method of aggregates and delivery of drugs and reagents Fig. 6 shows the process from the preparation of micro-aggregates carrying the drug to the release of the drug by dissociation.
To carry the drug on the microaggregates, the drug is added at the stage where the tannic acid derivative (PATA) is dissolved in a good solvent such as THF, and then the poor solvent is added by the method described above to form the microassociation. This is done by forming the coalescence in the liquid. At this stage, the microaggregates are encapsulated to protect the loaded drug. This microaggregate is stable even in water.
When the microaggregates are in a liquid such as water and are chemically stimulated by a surfactant, the bonds between the molecules of the membrane constituting the outer shell are broken and dissociated, and the inner air of the microaggregates. The drug carried on the part or the outer shell part is released into the liquid. Here, examples of the surfactant include CTAB (Cetyltrimethyl ammonium bromide), n-butylamine, n-hexylamine, diisopropylamine, triethylamine, aniline, tetra-n-butylammonium bromide and the like.
（実施例１）
実施例１では、本発明による微小会合体の製造と、その製造物の凝集性、大きさ、形状、外殻部の厚さについて調べた結果を述べる。(Example 1)
In Example 1, the production of the microaggregate according to the present invention and the results of investigating the cohesiveness, size, shape, and thickness of the outer shell portion of the product will be described.
＜微小会合体の製造＞
タンニン酸（ＴＡ、和光薬品株式会社製）と、その置換基となる１－ヨードヘキサン、１－ヨードデカンおよび１－ヨードヘキサデカン（全て東京化成工業製）とを準備した。そして、これらを原料とし、炭酸カリウム（和光薬品株式会社製）を触媒に用いたウィリアムソンエーテル合成法により、水酸基における水素原子のうち１０個が、炭素数が６、１０または１６のアルキル基で置換されたタンニン酸誘導体（ＰＡＴＡ）であるＴＡ（Ｃ６）１０、ＴＡ（Ｃ１０）１０およびＴＡ（Ｃ１６）１０をそれぞれ作製した。なお、この一連のタンニン酸誘導体作製工程は、アルゴン雰囲気下で行った。<Manufacturing of micro-aggregates>
Tannic acid (TA, manufactured by Wako Yakuhin Co., Ltd.) and 1-iodohexane, 1-iododecane and 1-iodohexadecane (all manufactured by Tokyo Chemical Industry Co., Ltd.) as substituents were prepared. Then, by the Williamson ether synthesis method using these as raw materials and using potassium carbonate (manufactured by Wako Yakuhin Co., Ltd.) as a catalyst, 10 of the hydrogen atoms in the hydroxyl group are alkyl groups having 6, 10 or 16 carbon atoms. Substituted tannin acid derivatives (PATA), TA (C 6 ) 10 , TA (C 10 ) 10 and TA (C 16 ) 10 , were prepared, respectively. This series of steps for producing a tannic acid derivative was carried out in an argon atmosphere.
次に、作製したタンニン酸誘導体を良溶媒であるＴＨＦに添加して良溶媒溶液を作製した。この良溶媒溶液の作製は、磁気スターラーを用いた攪拌下で行った。磁気スターラーの回転速度は３５０ｒｐｍとした。得られた良溶媒溶液を、ポアサイズ０．２μｍのメンブレンフィルターでろ過した。その後、ろ過された良溶媒溶液に、貧溶媒である水を、滴下ロートを用いて添加した。貧溶媒として用いた水は、抵抗率１８．２ＭΩでｐＨが６．８の超純水である。貧溶媒の添加中も、良溶媒溶液を作製するときと同様に、スターラーを用いて液を攪拌した。そして、溶液中のＴＨＦを２４時間かけて大気下で蒸発させ、その後、重力によるろ過を行って異物を取り除いた。この一連の会合体作製工程は室温（２５℃）下で行った。 Next, the prepared tannic acid derivative was added to THF as a good solvent to prepare a good solvent solution. The preparation of this good solvent solution was carried out under stirring using a magnetic stirrer. The rotation speed of the magnetic stirrer was 350 rpm. The obtained good solvent solution was filtered through a membrane filter having a pore size of 0.2 μm. Then, water, which is a poor solvent, was added to the filtered good solvent solution using a dropping funnel. The water used as the poor solvent is ultrapure water having a resistivity of 18.2 MΩ and a pH of 6.8. Even during the addition of the poor solvent, the liquid was stirred using a stirrer in the same manner as in preparing the good solvent solution. Then, THF in the solution was evaporated in the atmosphere for 24 hours, and then filtration was performed by gravity to remove foreign substances. This series of assembly preparation steps was performed at room temperature (25 ° C.).
＜微小会合体の形成確認および構造確認＞
最初に、微小会合体が形成されているかを光の散乱により確認した。
微小会合体形成工程後の水（ＰＡＴＡ ａｓｓｅｍｂｌｙ ｉｎ ｗａｔｅｒ）に光を照射したときの光散乱の様子（Ｂ）を、ＴＨＦ溶液中のタンニン酸誘導体（ＰＡＴＡ ｉｎ ＴＨＦ）に光を照射したときの光散乱の様子（Ａ）と比較して、図７に示す。微小会合体が形成された水の場合は、ＴＨＦ中のタンニン酸誘導体に比べ、光散乱による太めの光のパスが認められる。この光散乱の実験から、光を散乱する程度の大きさの会合体が形成されていることが確認された。<Confirmation of formation and structure of microaggregates>
First, it was confirmed by light scattering whether microaggregates were formed.
The state of light scattering when light is applied to water (PATA assembly in water) after the microaggregate forming step (B) is shown in the light when the tannic acid derivative (PATA in THF) in a THF solution is irradiated with light. It is shown in FIG. 7 in comparison with the state of scattering (A). In the case of water in which micro-aggregates are formed, a thicker light path due to light scattering is observed as compared with the tannic acid derivative in THF. From this light scattering experiment, it was confirmed that an aggregate large enough to scatter light was formed.
作製した微小会合体の大きさは、動的光散乱法（ＤＬＳ）および走査型電子顕微鏡（ＳＥＭ）により測定した。ＤＬＳの装置は、ＤＬＳ８０００ＨＡＬ（大塚電子製）である。同装置は、ＡＬＶ－ＳＰコンパクトゴニオメータ、ＡＬＶ５０００相互相関器およびＨｅＮｅレーザー（波長６３２．８ｎｍ）を備えている。ＳＥＭはＳＵ８０００（株式会社日立ハイテク製）であり、試料に白金をコートして１．０－１．５ｋＶの加速電圧で観察した。 The size of the prepared microaggregates was measured by dynamic light scattering (DLS) and scanning electron microscopy (SEM). The DLS device is DLS8000HAL (manufactured by Otsuka Electronics Co., Ltd.). The device is equipped with an ALV-SP compact goniometer, an ALV5000 cross-correlator and a HeNe laser (wavelength 632.8 nm). The SEM was SU8000 (manufactured by Hitachi High-Technologies Corporation), and the sample was coated with platinum and observed at an acceleration voltage of 1.0-1.5 kV.
ＤＬＳにより測定された微小会合体のゆらぎ信号強度分布と算出した粒径（流体力学的直径）Ｄｈの例を図８に示す。また、良溶媒に添加したタンニン酸誘導体の比率（濃度）をパラメータにしてＤＬＳ測定を行った結果を図９に示す。
その結果、微小会合体の直径（粒径）Ｄｈは、良溶媒に添加したタンニン酸誘導体の比率が０．０１ｍｇ／ｍＬから０．４０ｍｇ／ｍＬに高まるにつれて大きくなるが、０．４０ｍｇ／ｍＬでその傾向は飽和し、逆に１．２０ｍｇ／ｍＬになるとやや小さくなった。
良溶媒に添加したタンニン酸誘導体の比率（濃度）と、この微小会合体を含有する液体に波長３５０ｎｍの光を照射したときの透過率との関係を図１０に示す。凝集などによって大きな粒子が形成されると、その粒子によって光が散乱されて透過率が下がる。
図１０を見ると、濃度約０．２ｍｇ／ｍＬのところに変曲点がある。これは、図１１のＳＥＭ像からわかるように、会合体の凝集によるもので、濃度約０．２ｍｇ／ｍＬのところにあるＣＡＣ（Ｃｒｉｔｉｃａｌ Ａｇｇｒｅｇａｔｉｏｎ Ｃｏｎｃｅｎｔｒａｔｉｏｎ）より濃い濃度になると凝集体が形成される。孤立した会合体を形成するためには、良溶媒に添加するタンニン酸誘導体の濃度を０．２ｍｇ／ｍＬ以下とすることが求められる。
なお、図１０中には、この微小会合体の表面電荷密度を測定した例が併せて示されている。そのピークは約－５４ｍＶであり、これは、微小会合体の外側にタンニン酸の非置換芳香族水酸基が存在する傍証になっている。FIG. 8 shows an example of the fluctuation signal intensity distribution of the microaggregates measured by DLS and the calculated particle size (hydrodynamic diameter) Dh. Further, FIG. 9 shows the results of DLS measurement using the ratio (concentration) of the tannic acid derivative added to the good solvent as a parameter.
As a result, the diameter (particle size) Dh of the microaggregates increases as the ratio of the tannic acid derivative added to the good solvent increases from 0.01 mg / mL to 0.40 mg / mL, but at 0.40 mg / mL. The tendency was saturated, and conversely, it became slightly smaller at 1.20 mg / mL.
FIG. 10 shows the relationship between the ratio (concentration) of the tannic acid derivative added to the good solvent and the transmittance when the liquid containing the microaggregates is irradiated with light having a wavelength of 350 nm. When large particles are formed by aggregation or the like, the particles scatter light and the transmittance decreases.
Looking at FIG. 10, there is an inflection at a concentration of about 0.2 mg / mL. As can be seen from the SEM image of FIG. 11, this is due to the aggregation of aggregates, and aggregates are formed at a concentration higher than CAC (Critical Aggregation Concentration) at a concentration of about 0.2 mg / mL. In order to form an isolated aggregate, the concentration of the tannic acid derivative added to the good solvent is required to be 0.2 mg / mL or less.
In addition, an example of measuring the surface charge density of this microaggregate is also shown in FIG. Its peak is about -54 mV, which is evidence of the presence of an unsubstituted aromatic hydroxyl group of tannic acid outside the microaggregates.
図１２に、タンニン酸誘導体が添加された良溶媒溶液に添加する初期の貧溶媒（水）量と、ＤＬＳ測定により求めた粒径Ｄｈとの関係を示す。初期の水の添加量が６６体積％を境にして、粒径Ｄｈと粒径ばらつきの傾向は大きく異なる。水の添加量が６６体積％を下回る場合、粒径Ｄｈは１０００ｎｍレベルと大きく、そのばらつきも大きい。一方、７５体積％以上では、粒径Ｄｈは２００ｎｍレベルであり、その粒径ばらつきも小さなものとなっている。これは、図１３に示すＳＥＭ写真からわかるように、水の添加量が約７５体積％の場合は、孤立した会合体が形成されるのに対して、水の添加量が６６体積％を下回る場合には、会合体が凝集するためである。孤立した会合体を製造するには、初期に添加する貧溶媒（水）の量を６６体積％以上とすることが好ましく、７５体積％以上とすることがより好ましい。 FIG. 12 shows the relationship between the initial amount of poor solvent (water) added to the good solvent solution to which the tannic acid derivative is added and the particle size Dh determined by DLS measurement. When the initial amount of water added is 66% by volume, the particle size Dh and the tendency of the particle size variation are significantly different. When the amount of water added is less than 66% by volume, the particle size Dh is as large as 1000 nm, and the variation is also large. On the other hand, when the volume is 75% by volume or more, the particle size Dh is at the 200 nm level, and the variation in the particle size is small. As can be seen from the SEM photograph shown in FIG. 13, when the amount of water added is about 75% by volume, an isolated aggregate is formed, whereas the amount of water added is less than 66% by volume. In some cases, this is because the aggregates aggregate. In order to produce an isolated aggregate, the amount of the poor solvent (water) added at the initial stage is preferably 66% by volume or more, more preferably 75% by volume or more.
図１４に、タンニン酸誘導体が添加された良溶媒溶液に添加する貧溶媒（水）添加速度と、ＤＬＳ測定により求めた粒径Ｄｈとの関係を示す。水の添加速度が２ｍＬ／ｍｉｎ以上６００ｍＬ／ｍｉｎの範囲にわたって粒径Ｄｈが測定されており、会合体が形成されていることがわかる。特に、水の添加速度が２０ｍＬ／ｍｉｎ以上になると粒径Ｄｈのばらつきが小さくなり、水の添加速度の変化に対する粒径Ｄｈの変化は少なく、制御性が高くなる。図１５に、水の添加速度を変えて作製された微小会合体のＳＥＭ写真を示すが、水の添加速度が２ｍＬ／ｍｉｎのときは凝集体の生成率が高い。このことがＤＬＳ測定での粒径ばらつきの増大に繋がったと考えられる。なお、形成される微小会合体の形状は球状で、また、そのＳＥＭ観察された粒径（直径）は、ＤＬＳ測定による粒径Ｄｈと近いことが確認された。 FIG. 14 shows the relationship between the rate of addition of the poor solvent (water) added to the good solvent solution to which the tannic acid derivative was added and the particle size Dh determined by DLS measurement. The particle size Dh was measured over a range of water addition rate of 2 mL / min or more and 600 mL / min, indicating that aggregates were formed. In particular, when the water addition rate is 20 mL / min or more, the variation in the particle size Dh becomes small, the change in the particle size Dh with respect to the change in the water addition rate is small, and the controllability is improved. FIG. 15 shows an SEM photograph of microaggregates prepared by changing the water addition rate. When the water addition rate is 2 mL / min, the agglomerate formation rate is high. It is considered that this led to an increase in particle size variation in DLS measurement. It was confirmed that the shape of the formed microaggregates was spherical, and the particle size (diameter) observed by SEM was close to the particle size Dh measured by DLS.
次に、形成された微小会合体の外殻部の膜の厚さについて調べた。
最初に、小角Ｘ線散乱法（ＳＡＸＳ：ｓｍａｌｌ－ａｎｇｌｅ Ｘ－ｒａｙ ｓｃａｔｔｅｒｉｎｇ）により外殻部の厚さを試算した。ここで、ＳＡＸＳ測定は、銅のＫ－α線源を備えた測定装置であるＳＡＸＳｅｓｓ ｍｃ２（Ａｎｔｏｎ Ｐａａｒ製）の液体サンプル用標準キャピラリーセルを使用して実施した。厚さｄは、散乱ベクトルｑを用いて、２π／ｑで計算することができる。その結果、図１６に示すように、タンニン酸誘導体としてＴＡ（Ｃ１６）１０を用いた場合は厚さ５．２ｎｍ、ＴＡ（Ｃ１０）１０を用いた場合は厚さ３．４ｎｍになった。
図３に示すタンニン酸誘導体のリム部が内側に向かい、外側にタンニン酸の非置換水酸基部が露出している構造、図４に示すタンニン酸誘導体分子の大きさ、およびＳＡＸＳ法により求められた厚さを鑑みると、この微小会合体の外殻は、タンニン酸誘導体の２分子膜からなると考えられる。
また、透過型電子顕微鏡（ＴＥＭ）により、作製した微小会合体を観察した。その結果を図１７に示す。ＴＥＭとしては、ＪＥＭ－１０１０（ＪＥＯＬ製）を用い、加速電圧１００ｋＶで観察した。その結果、外殻部の厚さは約５ｎｍであり、上記ＳＡＸＳ法による測定と同様の値であった。Next, the thickness of the membrane in the outer shell of the formed microaggregates was investigated.
First, the thickness of the outer shell was estimated by the small-angle X-ray scattering method (SAXS). Here, the SAXS measurement was performed using a standard capillary cell for a liquid sample of SAXSess mc 2 (manufactured by Antonio Par), which is a measuring device equipped with a copper K-α radiation source. The thickness d can be calculated at 2π / q using the scattering vector q. As a result, as shown in FIG. 16, when TA (C 16 ) 10 was used as the tannic acid derivative, the thickness was 5.2 nm, and when TA (C 10 ) 10 was used, the thickness was 3.4 nm. ..
The structure in which the rim portion of the tannic acid derivative shown in FIG. 3 faces inward and the unsubstituted hydroxyl group portion of tannic acid is exposed to the outside, the size of the tannic acid derivative molecule shown in FIG. 4, and the SAXS method were used. Considering the thickness, the outer shell of this microaggregate is considered to be composed of a bilayer film of a tannic acid derivative.
In addition, the prepared micro-aggregates were observed with a transmission electron microscope (TEM). The result is shown in FIG. As a TEM, JEM-1010 (manufactured by JEOL) was used, and observation was performed at an acceleration voltage of 100 kV. As a result, the thickness of the outer shell portion was about 5 nm, which was the same value as the measurement by the SAXS method.
（実施例２）
実施例２は、親水性薬剤を担持した微小会合体の実施例を示す。
親水性薬剤（親水性プローブ）としてローダミンＢを用いて、ローダミンＢが微小会合体に担持されるかを調べた。
試料の作製方法は、タンニン酸誘導体（ＰＡＴＡ）を良溶媒であるＴＨＦに添加して良溶媒溶液を作製した段階で、ローダミンＢを添加する工程を追加し、他の工程は実施例１と同様に行い、微小会合体試料を作製した。ここで、ローダミンＢの添加量は５μＭとし、タンニン酸誘導体としてはＴＡ（Ｃ１６）１０を用いた。
そして、リファレンスとしてローダミンＢが添加されていない微小会合体も作製し、微小会合体の吸収スペクトル及び発光スペクトルについて、ローダミンＢ添加の有無による比較を行った。その結果を図１８に示すが、ローダミンＢを添加して微小会合体を作製したときに発光強度が大幅に低下しており、微小会合体に親水性プローブであるローダミンＢが担持されたことが確認された。ここで、発光スペクトルは、波長５２３ｎｍの光を試料に照射し、その蛍光をＦＰ－６６００（日本分光株式会社製）で測定して求めた。(Example 2)
Example 2 shows an example of a microaggregate carrying a hydrophilic agent.
Using rhodamine B as a hydrophilic agent (hydrophilic probe), it was investigated whether rhodamine B was supported on microaggregates.
As for the method for preparing a sample, a step of adding rhodamine B is added at the stage where a tannic acid derivative (PATA) is added to THF as a good solvent to prepare a good solvent solution, and the other steps are the same as in Example 1. To prepare a micro-aggregate sample. Here, the amount of rhodamine B added was 5 μM, and TA (C 16 ) 10 was used as the tannic acid derivative.
Then, as a reference, a microaggregate to which rhodamine B was not added was also prepared, and the absorption spectrum and the emission spectrum of the microaggregate were compared with and without the addition of rhodamine B. The results are shown in FIG. 18. It was found that the luminescence intensity was significantly reduced when the microaggregates were added to prepare the microaggregates, and that the microaggregates were supported by the hydrophilic probe rhodamine B. confirmed. Here, the emission spectrum was determined by irradiating the sample with light having a wavelength of 523 nm and measuring its fluorescence with FP-6600 (manufactured by JASCO Corporation).
（実施例３）
実施例３は、疎水性薬剤を担持した微小会合体の実施例を示す。
疎水性薬剤（疎水性プローブ）としてピレンを用いて、ピレンが微小会合体に担持されるかを調べた。
試料の作製方法は、実施例２と同じように、タンニン酸誘導体（ＰＡＴＡ）を良溶媒であるＴＨＦに添加して良溶媒溶液を作製した段階で、ピレンを添加する工程を追加し、他の工程は実施例１と同様に行い、微小会合体試料を作製した。ここで、ピレンの添加量は２μＭとし、タンニン酸誘導体としてはＴＡ（Ｃ１６）１０を用いた。
そして、リファレンスとして、タンニン酸誘導体に代えて同モル量のタンニン酸を用いた試料、およびタンニン酸誘導体を添加しないで、良溶媒とピレンとからなる溶液に水のみが添加された試料も作製し、その発光スペクトルの比較を行った。その結果を図１９に示すが、タンニン酸誘導体を用いたときに明確な発光スペクトルが観測され、微小会合体に疎水性プローブであるピレンが担持されたことが確認された。ここで、発光スペクトルは、波長３９９ｎｍの光を試料に照射し、その蛍光を測定したものである。(Example 3)
Example 3 shows an example of a microaggregate carrying a hydrophobic agent.
Using pyrene as a hydrophobic agent (hydrophobic probe), it was investigated whether pyrene was supported on microaggregates.
As for the method for preparing a sample, as in Example 2, a step of adding pyrene is added at the stage where a tannic acid derivative (PATA) is added to THF as a good solvent to prepare a good solvent solution, and another step is added. The step was the same as in Example 1 to prepare a microaggregate sample. Here, the amount of pyrene added was 2 μM, and TA (C 16 ) 10 was used as the tannic acid derivative.
Then, as a reference, a sample using the same molar amount of tannic acid instead of the tannic acid derivative and a sample in which only water was added to a solution consisting of a good solvent and pyrene without adding the tannic acid derivative were also prepared. , The emission spectra were compared. The results are shown in FIG. 19, and a clear emission spectrum was observed when the tannic acid derivative was used, confirming that pyrene, which is a hydrophobic probe, was supported on the microaggregates. Here, the emission spectrum is obtained by irradiating a sample with light having a wavelength of 399 nm and measuring its fluorescence.
（実施例４）
実施例４は、親水性プローブが担持された微小会合体の化学的刺激による解離の実施例を示す。
実施例２により作製した、親水性プローブとしてのローダミンＢが担持された微小会合体を有する水に、化学的刺激剤であるＣＴＡＢを添加して、化学的刺激剤による微小会合体の解離をスペクトル測定およびＳＥＭ観察により調べた。(Example 4)
Example 4 shows an example of chemical-stimulated dissociation of microaggregates carrying a hydrophilic probe.
CTAB, which is a chemical stimulant, is added to water having microaggregates carrying rhodamine B as a hydrophilic probe prepared in Example 2, and the dissociation of the microaggregates by the chemical stimulant is spectrally observed. It was examined by measurement and SEM observation.
図２０は、ＣＴＡＢを添加したときの発光強度スペクトルを、添加後の経過時間をパラメータにして示した特性図である。ここで、この実験は、入射光の波長を５２３ｎｍとしたときの蛍光スペクトルをＦＰ－６６００（日本分光株式会社製）でモニタした結果である。
ＣＴＡＢ添加後の時間が進むほど、ローダミンＢを示す波長約５８０ｎｍの発光強度は大幅に上昇し、微小会合体が解離してローダミンＢが液中に放出されていることが分かる。ここで、ＣＴＡＢはカチオン性の界面活性剤であり、その添加量は０．４ｍＭで、その量は臨界ミセル濃度（ＣＭＣ）の約０．９ｍＭの半分以下である。
図２１は、ＣＴＡＢを添加した場合と添加しない場合とで、発光強度の時間変化を比較した特性図である。発光強度は、ローダミンＢを特徴づける波長５８０ｎｍでの光強度とした。したがって、発光強度が高いほどローダミンＢが放出されていることを示す。ここで、ＣＴＡＢの添加量は、図２０の場合と同様に０．４ｍＭである。
ＣＴＡＢを添加した場合は、時間とともに発光強度が上昇し、微小会合体が解離して担持剤（模擬担持薬剤）であるローダミンＢが液中に放出されていることが分かる。一方で、ＣＴＡＢを添加しない場合は、発光強度に有意な時間変化は認められず、ローダミンＢが微小会合体に担持された状態を維持していることがわかる。FIG. 20 is a characteristic diagram showing the emission intensity spectrum when CTAB is added, with the elapsed time after the addition as a parameter. Here, this experiment is the result of monitoring the fluorescence spectrum when the wavelength of the incident light is 523 nm with FP-6600 (manufactured by JASCO Corporation).
It can be seen that as the time after the addition of CTAB progresses, the emission intensity at a wavelength of about 580 nm, which indicates rhodamine B, increases significantly, and the microaggregates dissociate and rhodamine B is released into the liquid. Here, CTAB is a cationic surfactant, the amount of which is 0.4 mM, which is less than half of the critical micelle concentration (CMC) of about 0.9 mM.
FIG. 21 is a characteristic diagram comparing changes in emission intensity over time between the case where CTAB is added and the case where CTAB is not added. The emission intensity was the light intensity at a wavelength of 580 nm, which characterizes Rhodamine B. Therefore, the higher the emission intensity, the more rhodamine B is released. Here, the amount of CTAB added is 0.4 mM as in the case of FIG. 20.
It can be seen that when CTAB is added, the luminescence intensity increases with time, the microaggregates dissociate, and rhodamine B, which is a carrier (simulated carrier), is released into the liquid. On the other hand, when CTAB was not added, no significant change in luminescence intensity was observed, indicating that rhodamine B was maintained on the microaggregates.
図２２は、薬剤を親水性模擬薬剤であるローダミンＢとしたときの、無担持（薬剤無内包）、担持（親水性薬剤（ローダミンＢ）内包担持）およびＣＴＡＢによる部分解離（会合体部分解離）の各状態でのＳＥＭ写真を示す。
ローダミンＢが担持された状態では、微小会合体の内部が黒化しており、ローダミンＢが内包されていることが分かる。この状態では、微小会合体の外部はローダミンＢが担持されていない微小会合体の外部と同じであり、微小会合体の外部へのパーティクル放出は認められない。
一方、ＣＴＡＢが添加された場合は、左側半分の領域において外殻部境界のコントラストが低下し、微小会合体の部分解離が起こっていることが分かる。さらに、微小会合体の外部には多数の微小なパーティクルが認められる。このパーティクルは、微小会合体に内包されていたローダミンＢが放出されたことによるものと考えられる。
以上から、親水性の薬剤が担持された微小会合体に適当な化学的刺激を与えることにより、微小会合体は解離し、親水性の薬剤を放出、供与できることが確認された。FIG. 22 shows, when the drug is Rhodamine B, which is a hydrophilic simulated drug, no support (drug-free inclusion), support (hydrophilic drug (Rhodamine B) inclusion support), and partial dissociation by CTAB (aggregate partial dissociation). The SEM photograph in each state of is shown.
In the state where rhodamine B is supported, the inside of the microaggregate is blackened, and it can be seen that rhodamine B is contained. In this state, the outside of the microaggregate is the same as the outside of the microaggregate on which rhodamine B is not supported, and no particle emission to the outside of the microaggregate is observed.
On the other hand, when CTAB was added, the contrast at the outer shell boundary decreased in the region on the left half, indicating that partial dissociation of the microaggregates occurred. Furthermore, a large number of fine particles are found outside the microaggregates. It is probable that these particles were due to the release of rhodamine B contained in the microaggregates.
From the above, it was confirmed that by giving an appropriate chemical stimulus to the microaggregates carrying the hydrophilic drug, the microaggregates dissociate and the hydrophilic drug can be released and donated.
（実施例５）
実施例５は、疎水性薬剤が担持された微小会合体の化学的刺激による解離の実施例を示す。
そこでは、実施例３により作製した、疎水性プローブとしてのピレンが担持された微小会合体が混入された水に、化学的刺激剤としてＣＴＡＢを添加して、化学的刺激剤による微小会合体の解離を発光スペクトル測定により調べた。但し、実施例５ではピレンの添加量は１μＭとした。(Example 5)
Example 5 shows an example of chemical-stimulated dissociation of microaggregates carrying a hydrophobic agent.
There, CTAB was added as a chemical stimulant to the water in which the microaggregate carrying pyrene as a hydrophobic probe was mixed, which was prepared in Example 3, and the microaggregate was prepared by the chemical stimulant. Dissociation was examined by emission spectrum measurement. However, in Example 5, the amount of pyrene added was 1 μM.
図２３は、ＣＴＡＢを添加したときの発光強度スペクトルを、ＣＴＡＢの添加量をパラメータにして示した特性図である。ここで、測定はＣＴＡＢ添加後６時間経過した時点で行われた。波長３９９ｎｍの光を入射させて、その蛍光スペクトルを測定した。測定器は実施例４で用いたものと同じである。
ＣＴＡＢ添加の添加量が多いほど、ピレンが外殻部に担持された膜を示す発光スペクトルの強度が下がり、微小会合体の解離が起こっていることがわかる。微小会合体が解離して放出されたピレンの量をこの図から計算すると、０．０５ｍＭのＣＴＡＢを添加した場合が約４１％であり、０．６ｍＭのＣＴＡＢを添加した場合が約８４％である。FIG. 23 is a characteristic diagram showing the emission intensity spectrum when CTAB is added, with the amount of CTAB added as a parameter. Here, the measurement was performed 6 hours after the addition of CTAB. Light having a wavelength of 399 nm was incident and the fluorescence spectrum was measured. The measuring instrument is the same as that used in Example 4.
It can be seen that the larger the amount of CTAB added, the lower the intensity of the emission spectrum showing the film on which pyrene is supported on the outer shell, and the dissociation of microaggregates occurs. When the amount of pyrene released by dissociating the microaggregates was calculated from this figure, it was about 41% when 0.05 mM CTAB was added and about 84% when 0.6 mM CTAB was added. be.
次に、化学的刺激剤の種類を変えて蛍光発光量の添加濃度依存性を測定することにより、ピレンが担持された微小会合体の解離特性を調べた。その結果を図２４および図２５にそれぞれ示す。ここで、用いた化学的刺激剤は、カチオン性界面活性剤のＣＴＡＢ、アニオン性の界面活性剤ＳＤＳ（Ｓｏｄｉｕｍ Ｄｏｄｅｃｙｌ Ｓｕｌｆａｔｅ）、非イオン性の界面活性剤Ｔｒｉｔｏｎ Ｘ－１００（２９－（４－ｔｅｒｔ－Ｏｃｔｙｌｐｈｅｎｏｘｙ）－３，６，９，１２，１５，１８，２１，２４，２７－ｎｏｎａｏｘａｎｏｎａｃｏｓａｎ－１－ｏｌ）、Ｔｗｅｅｎ４０（Ｐｏｌｙｏｘｙｅｔｈｙｌｅｎｅ ｓｏｒｂｉｔａｎ ｍｏｎｏｐａｌｍｉｔａｔｅ）、アミン類（ｎ－Ｂｕｔｈｙｌａｍｉｎｅ、ｎ－Ｈｅｘｙｌａｍｉｎｅ、Ｄｉｉｓｏｐｒｏｐｙｌａｍｉｎｅ、Ｔｒｉｅｔｈｙｌａｍｉｎｅ、ＡｎｉｌｉｎｅおよびＭｅｔｈｏｘｙｐｏｌｙｅｔｈｙｌｅｎｅ ｇｌｙｃｏｌ ａｍｉｎｅ）、１－ＨｅｘａｎｏｌならびにＴｅｔｒａ－ｎ－ｂｕｔｈｙｌａｍｍｏｎｉｕｍ ｂｒｏｍｉｄｅである。なお、この測定は化学的刺激剤を添加した６時間後に測定した。また、参考までに、各実験の発光スペクトルを図２６から図３１にそれぞれ示す。
その結果、カチオン性界面活性剤およびアミンを化学的刺激剤とした場合は、添加濃度の増加による発光量の有意な減少が認められ、微小会合体が解離して、担持されていたピレンが放出されたことが確認された。一方、アニオン性界面活性剤および非イオン性界面活性剤を化学的刺激剤としたときは、添加濃度の増加による発光量の減少は僅かであり、有意な減少があるとは認められなかった。すなわち、アニオン性界面活性剤および非イオン性界面活性剤を化学的刺激剤としたときは、微小会合体の有意な解離は認められなかった。この理由は、微小会合体のゼータ電位がマイナスであるため、カチオン性界面活性剤は静電的に引き寄せられるが、アニオン性またはノニオン性の界面活性剤に対しては静電相互作用が働かないためであると考えられる。なお、図２６（ｂ）、図２７（ａ）、図２７（ｂ）および図３０（ａ）においては、濃度依存性が小さいため、スペクトル特性曲線に重なりが認められる。
以上から、微小会合体に疎水性の薬剤を担持させたときも、適当な化学的刺激を与えることにより、微小会合体は解離し、疎水性の薬剤を放出、供与できることが確認された。Next, the dissociation characteristics of the microaggregates carrying pyrene were investigated by measuring the addition concentration dependence of the amount of fluorescence emission by changing the type of the chemical stimulant. The results are shown in FIGS. 24 and 25, respectively. Here, the chemical stimulants used were CTAB, a cationic surfactant, SDS (Sodium Dodecyl Sulfate), an anionic surfactant, and Triton X-100 (29- (4-tert), a nonionic surfactant. -Octylphenoxy) -3,6,9,12,15,18,21,24,27-nonanoxanonacosan-1-ol), Tween40 (Polyoxythelylene sorbitan monopalmate), amines (n-Buthylamine, n-Hexylamine, n-Hexylamine) , Aniline and Chemoxypolythethylene glycol amine), 1-Hexanol and Triton-n-buthylammonium bromide. This measurement was performed 6 hours after the addition of the chemical stimulant. For reference, the emission spectra of each experiment are shown in FIGS. 26 to 31, respectively.
As a result, when a cationic surfactant and an amine were used as chemical stimulants, a significant decrease in the amount of luminescence was observed due to an increase in the added concentration, the microaggregates were dissociated, and the carried pyrene was released. It was confirmed that it was done. On the other hand, when the anionic surfactant and the nonionic surfactant were used as chemical stimulants, the decrease in the amount of luminescence due to the increase in the added concentration was slight, and no significant decrease was observed. That is, when anionic surfactant and nonionic surfactant were used as chemical stimulants, no significant dissociation of microaggregates was observed. The reason for this is that the zeta potential of the microaggregates is negative, so that the cationic surfactant is electrostatically attracted, but the electrostatic interaction does not work with the anionic or nonionic surfactant. It is thought that this is the reason. In FIGS. 26 (b), 27 (a), 27 (b), and 30 (a), since the concentration dependence is small, overlap is observed in the spectral characteristic curves.
From the above, it was confirmed that even when a hydrophobic drug is supported on the microaggregates, the microaggregates can be dissociated and the hydrophobic drug can be released and donated by applying an appropriate chemical stimulus.
本発明により、人体、環境に馴染みやすく悪影響が少ないと考えられる天然植物由来の材料を用いた微小会合体を提供することが可能になる。
そして、この微小会合体は、薬剤等を担持させることができ、界面活性剤による化学的刺激により容易に解体させることができるので、薬剤、酵素、試薬等のキャリアとして優れた効果を発揮する能力をもつ。
しかも、この微小会合体は、親水性、疎水性どちらの薬剤等も担持可能である。さらに、その微小会合体は、生体等への適用に適した１００ｎｍ以上２００ｎｍ以下のナノ領域の大きさであり、自己組織化形成で製造できるため、その会合体の製造効率も高い。
以上のことから、本発明の微小会合体は、医療用を始めとして、バイオ燃料電池、バイオセンサー、エネルギー変換素子、分析法など様々な分野への適用が期待される。INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible to provide a microaggregate using a material derived from a natural plant, which is considered to be easily compatible with the human body and the environment and has little adverse effect.
Since this microaggregate can carry a drug or the like and can be easily disassembled by chemical stimulation with a surfactant, it has an ability to exert an excellent effect as a carrier of a drug, an enzyme, a reagent or the like. Has.
Moreover, the microaggregates can carry both hydrophilic and hydrophobic agents. Further, the micro-aggregate has a size of a nano region of 100 nm or more and 200 nm or less suitable for application to a living body or the like, and can be produced by self-organization formation, so that the production efficiency of the aggregate is also high.
From the above, the microaggregate of the present invention is expected to be applied to various fields such as biofuel cells, biosensors, energy conversion elements, and analytical methods, including medical use.
Claims (14)
前記殻構造は、タンニン酸の少なくとも一部の水酸基における水素原子が直鎖の炭化水素基で置換されたタンニン酸誘導体の２分子膜構造体からなり、
前記直鎖の炭化水素基の炭素数が６以上１６以下である、微小会合体。 The outermost is a microaggregate forming a shell structure,
The shell structure consists of a bilayer film structure of a tannic acid derivative in which hydrogen atoms at at least a part of the hydroxyl groups of tannic acid are replaced with linear hydrocarbon groups.
A microaggregate having 6 or more and 16 or less carbon atoms in the linear hydrocarbon group .
前記タンニン酸誘導体を良溶媒に溶解させる溶解工程と、
前記タンニン酸誘導体が溶解した良溶媒に貧溶媒を添加する貧溶媒添加工程とを有する、
請求項１から１１の何れか１に記載の微小会合体を製造する微小会合体製造方法。 A tannic acid derivative preparation step for preparing a tannic acid derivative in which a hydrogen atom in at least a part of the hydroxyl groups of tannic acid is replaced with a linear hydrocarbon group, and a step of preparing the tannic acid derivative.
A dissolution step of dissolving the tannic acid derivative in a good solvent, and
It comprises a poor solvent addition step of adding a poor solvent to a good solvent in which the tannic acid derivative is dissolved.
The method for producing a microaggregate according to any one of claims 1 to 11 .
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