JP2006028069A - Method for producing liposome utilizing coacervate - Google Patents
Method for producing liposome utilizing coacervate Download PDFInfo
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- JP2006028069A JP2006028069A JP2004207860A JP2004207860A JP2006028069A JP 2006028069 A JP2006028069 A JP 2006028069A JP 2004207860 A JP2004207860 A JP 2004207860A JP 2004207860 A JP2004207860 A JP 2004207860A JP 2006028069 A JP2006028069 A JP 2006028069A
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Abstract
Description
本発明は、リポソームの製造方法に関し、特に、糖脂質系バイオサーファクタント化合物や生理活性成分を含むコアセルベートから、熱力学的に安定かつ高濃度に生理活性成分を含有したリポソームを製造する方法、該製造方法により製造されたリポソーム、およびこれらを配合した医薬品等に関する。 The present invention relates to a method for producing liposomes, and in particular, a method for producing liposomes containing a bioactive component in a thermodynamically stable and high concentration from a coacervate containing a glycolipid biosurfactant compound and a bioactive component, and the production The present invention relates to liposomes produced by the method, and pharmaceuticals containing these.
リポソームは脂質二分子膜を構成単位とする閉鎖小胞体であり、生体膜との親和性が高いことから、内部の水相や二分子膜内部に種々の薬剤等の生物活性物質を含有させ、それらの安定性の改善、活性の持続性の改善、及び標的組織への到達性の増大を図る試みが行われている。また最近では、上記のような生物活性物質をコードする遺伝子をリポソームと混合して複合体を作り、またはリポソームに封入保持し、これを患者に投与することにより標的組織の細胞内に遺伝子を注入し、生物活性物質をその場で生産させる遺伝子治療が研究されている。特に、がんの治療では、がんを抑制する遺伝子(アンチセンスDNAなど)を体内注入する手法が、化学療法に代わる新しい治療法として注目されている。リポソームへ薬剤、生理活性物質、遺伝子を簡便に高効率で含有させるためには、超臨界炭酸ガスを使用するリポソーム製造法(例えば、特許文献1参照)などが報告されているが、多大なエネルギーやコストを消費するため実用化には適していない。 Liposomes are closed endoplasmic reticulums with lipid bilayers as structural units, and because they have high affinity with biological membranes, they contain biologically active substances such as various drugs in the internal aqueous phase and bilayer membranes. Attempts have been made to improve their stability, improve activity persistence, and increase reachability to target tissues. Recently, a gene encoding a biologically active substance as described above is mixed with a liposome to form a complex, or the liposome is encapsulated and retained, and then administered to a patient to inject the gene into the cells of the target tissue. However, gene therapy that produces bioactive substances on the spot has been studied. In particular, in the treatment of cancer, a technique of injecting a gene that suppresses cancer (antisense DNA or the like) into the body is attracting attention as a new treatment method replacing chemotherapy. In order to easily contain drugs, physiologically active substances, and genes in liposomes with high efficiency, a liposome production method using supercritical carbon dioxide (for example, see Patent Document 1) has been reported. It is not suitable for practical use because of the cost and cost.
また、一般にリポソームは水溶液の状態では熱力学的に不安定であり、リポソーム粒子同士の凝集や融合、膜成分の結晶化による沈澱の生成、粒子径の増大などがおこり、効力及び外観変化による商品価値の損失となりやすかった。リポソームの水性懸濁液の安定化については検討例が少ないが、その方法として多価アルコール及びまたは糖類を配合して安定化させる方法が報告されている(例えば、特許文献2参照)。また、アミノ酸を用いてリポソームを安定化する方法も報告されている(例えば、特許文献3参照)。
しかしながら、上記方法で解決したのは室温以下の保存温度での安定性のみであり、室温においても水溶液状態で長期間安定なリポソームを提供するものではない。さらに、リポソームを用いる生体組織、細胞への物質導入には必要不可欠なリポソームへの薬剤、生理活性物質、遺伝子等を簡便に高効率で含有させる方法についても解決すべき問題が多く、実用化されていない。
In general, liposomes are thermodynamically unstable in the state of an aqueous solution, causing aggregation and fusion of liposome particles, formation of precipitates due to crystallization of membrane components, increase in particle diameter, etc., and products resulting from changes in efficacy and appearance. It was easy to lose value. There are few studies on the stabilization of aqueous suspensions of liposomes, but a method of stabilizing by mixing polyhydric alcohol and / or saccharide has been reported (for example, see Patent Document 2). A method for stabilizing liposomes using amino acids has also been reported (see, for example, Patent Document 3).
However, only the stability at the storage temperature below room temperature has been solved by the above method, and it does not provide a liposome that is stable for a long time in an aqueous solution state even at room temperature. Furthermore, there are many problems that need to be solved with regard to methods for easily and efficiently containing drugs, physiologically active substances, genes, etc. in liposomes that are indispensable for the introduction of substances into living tissues and cells using liposomes. Not.
従って、本発明の目的は、薬物、生理活性物質、遺伝子などの物質を効率良く封入でき、標的細胞へ高い効率で導入でき、さらには、水溶液状態で長時間安定なリポソームの製造方法、該製造方法により製造されるリポソーム、およびそれよりなる物質導入担体を提供することにある。 Therefore, an object of the present invention is to efficiently encapsulate substances such as drugs, physiologically active substances, and genes, to introduce them into target cells with high efficiency, and to produce a liposome that is stable in an aqueous solution for a long period of time. An object of the present invention is to provide a liposome produced by the method, and a substance introduction carrier comprising the liposome.
上記したような従来技術の欠点を克服し、リポソームに薬物、生理活性物質、遺伝子などの物質を効率良く封入でき、かつ室温において水溶液状態で長期間安定なリポソームの製造方法および該リポソームについて鋭意研究を行った結果、糖脂質を用いて形成したコアセルベートから、リポソームを形成せしめることによって、薬物、生理活性物質、遺伝子などの物質を効率良く封入でき、かつ水溶液状態で長時間安定なリポソームの製造方法および該リポソームを見出し、本発明を完成させたものである。 Overcoming the drawbacks of the prior art as described above, and capable of efficiently encapsulating substances such as drugs, physiologically active substances and genes in liposomes, and producing a liposome that is stable in an aqueous solution at room temperature for a long period of time, and earnest research on the liposome As a result, by forming liposomes from coacervates formed using glycolipids, it is possible to efficiently encapsulate substances such as drugs, physiologically active substances and genes, and to produce liposomes that are stable in aqueous solution for a long time The present inventors have found the liposome and completed the present invention.
本発明の課題は以下の手段によって達成される。
(1)糖脂質を用いて形成したコアセルベートより調製することを特徴とするリポソームの製造方法。
(2)前記糖脂質が下記一般式1で表されるマンノシドリピド系化合物であることを特徴とする、上記(1)記載のリポソームの製造方法。
The object of the present invention is achieved by the following means.
(1) A method for producing a liposome, which is prepared from a coacervate formed using a glycolipid.
(2) The method for producing a liposome according to (1) above, wherein the glycolipid is a mannoside lipid compound represented by the following general formula 1.
上記一般式1中、R11、R12、R13、R14、R15、R16、R17およびR18は、それぞれ水素原子、CH2(OH)−〔CH(OH)〕m1−CH2−基、飽和もしくは不飽和で直鎖もしくは分岐鎖の脂肪族炭化水素基、または脂肪族アシル基を示す。m1は0〜8の整数を示す。またn1は0〜10の整数を示す。(ただし、R11、R12、R13、R14、R15、R16、R17およびR18の全てが水素原子である場合を除く。)
(3)前記マンノシドリピド系化合物が、下記一般式2で表される化合物であることを特徴とする、上記(2)記載のリポソームの製造方法。
In the general formula 1, R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 and R 18 are each a hydrogen atom, CH 2 (OH) — [CH (OH)] m1 —CH A 2 -group, a saturated or unsaturated, linear or branched aliphatic hydrocarbon group, or an aliphatic acyl group. m1 represents an integer of 0 to 8. N1 represents an integer of 0 to 10. (However, the case where all of R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 and R 18 are hydrogen atoms is excluded.)
(3) The method for producing a liposome according to the above (2), wherein the mannoside lipid compound is a compound represented by the following general formula 2.
上記一般式2中、R21、R22、R23、R24およびR25は、それぞれ水素原子、CH2(OH)−〔CH(OH)〕m2−CH2−基、飽和もしくは不飽和で直鎖もしくは分岐鎖の脂肪族炭化水素基、または脂肪族アシル基を示す。また、m2は0〜8の整数を示す。(ただし、R21、R22、R23、R24およびR25の全てが水素原子である場合を除く。)
(4)前記糖脂質が下記一般式3または4で表されるラムノースリピド系化合物であることを特徴とする、上記(1)記載のリポソームの製造方法。
In the above general formula 2, R 21 , R 22 , R 23 , R 24 and R 25 are each a hydrogen atom, CH 2 (OH) — [CH (OH)] m 2 —CH 2 — group, saturated or unsaturated. A linear or branched aliphatic hydrocarbon group or an aliphatic acyl group is shown. Moreover, m2 shows the integer of 0-8. (However, the case where all of R 21 , R 22 , R 23 , R 24 and R 25 are hydrogen atoms is excluded.)
(4) The method for producing a liposome according to the above (1), wherein the glycolipid is a rhamnose lipid compound represented by the following general formula 3 or 4.
上記一般式3中、R33およびR34は、それぞれ水素原子、飽和もしくは不飽和で直鎖もしくは分岐鎖の脂肪族炭化水素基、脂肪族アシル基、または下記一般式5で表される基を示す。また、m3およびn3は、それぞれ1〜30の整数である。 In the general formula 3, R 33 and R 34 are each a hydrogen atom, a saturated or unsaturated, linear or branched aliphatic hydrocarbon group, an aliphatic acyl group, or a group represented by the following general formula 5. Show. M3 and n3 are each an integer of 1 to 30.
上記一般式4中、R41およびR42は、それぞれ水素原子、飽和もしくは不飽和で直鎖もしくは分岐鎖の脂肪族炭化水素基、脂肪族アシル基、または下記一般式5で表される基を示す。また、m4は1〜30の整数である。 In the general formula 4, R 41 and R 42 are each a hydrogen atom, a saturated or unsaturated, linear or branched aliphatic hydrocarbon group, an aliphatic acyl group, or a group represented by the following general formula 5. Show. Moreover, m4 is an integer of 1-30.
上記一般式5中、n5は1〜30の整数である。
(5)前記糖脂質が下記一般式6または7で表されるソフォロースリピド系化合物であることを特徴とする、上記(1)記載のリポソームの製造方法。
In the general formula 5, n5 is an integer of 1-30.
(5) The method for producing a liposome according to (1) above, wherein the glycolipid is a Sofollows lipid compound represented by the following general formula 6 or 7.
上記一般式6中、R61およびR62は、それぞれ水素原子、飽和もしくは不飽和で直鎖もしくは分岐鎖の脂肪族炭化水素基、または脂肪族アシル基を示し、m6は1〜30の整数を示す。 In the general formula 6, R 61 and R 62 each represent a hydrogen atom, a saturated or unsaturated, linear or branched aliphatic hydrocarbon group, or an aliphatic acyl group, and m6 represents an integer of 1 to 30. Show.
上記一般式7中、R71およびR72は、それぞれ水素原子、飽和もしくは不飽和で直鎖もしくは分岐鎖の脂肪族炭化水素基、または脂肪族アシル基を示し、R73は水素原子、または飽和もしくは不飽和で直鎖もしくは分岐鎖の脂肪族炭化水素基を示し、m7は1〜30の整数を示す。
(6)前記糖脂質が下記一般式8で表されるトレハロースリピド系化合物であることを特徴とする、上記(1)記載のリポソームの製造方法。
In the general formula 7, R 71 and R 72 each represent a hydrogen atom, a saturated or unsaturated, linear or branched aliphatic hydrocarbon group, or an aliphatic acyl group, and R 73 represents a hydrogen atom or a saturated group. Alternatively, it represents an unsaturated linear or branched aliphatic hydrocarbon group, and m7 represents an integer of 1 to 30.
(6) The method for producing a liposome according to (1) above, wherein the glycolipid is a trehalose lipid compound represented by the following general formula 8.
上記一般式8中、R81およびR82は、水素原子、下記一般式9で表される基、または下記一般式10で表される基を示す。ただし、R81及びR82がともに水素原子である場合を除く。 In General Formula 8, R 81 and R 82 represent a hydrogen atom, a group represented by General Formula 9 below, or a group represented by General Formula 10 below. However, the case where both R 81 and R 82 are hydrogen atoms is excluded.
上記一般式9中、m9≧14、n9≧13、m9+n9=1〜50である。また、一般式9で表される基には、2重結合、分岐、ケトン基、シクロプロパン環、メトキシル基が含まれることがある。 In the general formula 9, m9 ≧ 14, n9 ≧ 13, and m9 + n9 = 1 to 50. The group represented by the general formula 9 may include a double bond, a branch, a ketone group, a cyclopropane ring, and a methoxyl group.
上記一般式10中、m10≧14、n10≧13、m10+n10=1〜50である。また、一般式10で表される基には、2重結合、分岐、ケトン基、シクロプロパン環、メトキシル基が含まれることがある。
(7)前記糖脂質が下記一般式11で表されるサクシノイルトレハロースリピド系化合物であることを特徴とする、上記(1)記載のリポソームの製造方法。
In the general formula 10, m10 ≧ 14, n10 ≧ 13, and m10 + n10 = 1 to 50. In addition, the group represented by the general formula 10 may include a double bond, a branch, a ketone group, a cyclopropane ring, and a methoxyl group.
(7) The method for producing a liposome according to (1) above, wherein the glycolipid is a succinoyl trehalose lipid compound represented by the following general formula 11.
上記一般式11中、R111、R112、R113およびR114は、それぞれ水素原子または脂肪族アシル基を示し、かつR111、R112、R113およびR114のうち少なくとも1つはサクシノイル基である。
(8)前記糖脂質が下記一般式12で表されるセロビオースリピド系化合物であることを特徴とする、上記(1)記載のリポソームの製造方法。
In the general formula 11, R 111 , R 112 , R 113 and R 114 each represent a hydrogen atom or an aliphatic acyl group, and at least one of R 111 , R 112 , R 113 and R 114 is a succinoyl group. It is.
(8) The method for producing a liposome according to the above (1), wherein the glycolipid is a cellobiose lipid compound represented by the following general formula 12.
上記一般式12中、R121は水素原子またはOHを示し、R122、R123およびR124は、それぞれ水素原子、飽和もしくは不飽和で直鎖もしくは分岐鎖の脂肪族炭化水素基、脂肪族アシル基、または下記一般式13で表される基を示し、m12は1〜30の整数を示す。 In the general formula 12, R 121 represents a hydrogen atom or OH, and R 122 , R 123 and R 124 each represents a hydrogen atom, a saturated or unsaturated, linear or branched aliphatic hydrocarbon group, and an aliphatic acyl. A group or a group represented by the following general formula 13; and m12 represents an integer of 1 to 30.
上記一般式13中、n13は1〜30の整数を示す。
(9)前記糖脂質が下記一般式14で表されるグルコシドリピド系化合物であることを特徴とする、上記(1)記載のリポソームの製造方法。
In the general formula 13, n13 represents an integer of 1-30.
(9) The method for producing a liposome according to (1), wherein the glycolipid is a glucosidolipid compound represented by the following general formula 14.
上記一般式14中、R141、R142、R143、R144、R145、R146、R147およびR148は、それぞれ水素原子、CH2(OH)−〔CH(OH)〕m14−CH2−基、飽和もしくは不飽和で直鎖もしくは分岐鎖の脂肪族炭化水素基、または脂肪族アシル基を示し、m14は0〜8の整数を示す。n14は0〜10の整数を示す。(ただし、R141、R142、R143、R144、R145、R146、R147およびR148の全てが水素原子である場合を除く。)
(10)前記糖脂質が下記一般式15で表されるアルカノイル−N−メチルグルカミドリピド系化合物であることを特徴とする、上記(1)記載のリポソームの製造方法。
In the general formula 14, R 141 , R 142 , R 143 , R 144 , R 145 , R 146 , R 147 and R 148 are each a hydrogen atom, CH 2 (OH) — [CH (OH)] m14 —CH 2- group, a saturated or unsaturated, linear or branched aliphatic hydrocarbon group, or an aliphatic acyl group, m14 represents an integer of 0-8. n14 represents an integer of 0 to 10. (However, the case where R 141 , R 142 , R 143 , R 144 , R 145 , R 146 , R 147 and R 148 are all hydrogen atoms is excluded.)
(10) The method for producing a liposome according to (1) above, wherein the glycolipid is an alkanoyl-N-methylglucamide lipid compound represented by the following general formula 15.
上記一般式15中、R151は飽和または不飽和で直鎖または分岐鎖の脂肪族炭化水素基を示す。
(11)前記糖脂質が、糖脂質系バイオサーファクタント化合物であることを特徴とする、上記(1)〜(10)のいずれかに記載のリポソームの製造方法。
(12)形成したコアセルベートを構成する糖脂質の化学構造を変化させて調製することを特徴とする、上記(1)〜(11)のいずれかに記載のリポソームの製造方法。
(13)形成したコアセルベートに、リン脂質、もしくは該コアセルベートを形成する糖脂質以外の糖脂質を混合することによって、調製することを特徴とする、上記(1)〜(11)のいずれかに記載のリポソームの製造方法。
(14)形成したコアセルベートに、電気的刺激を与えることにより、または、圧力もしくは温度を変化せしめることによって、調製することを特徴とする、上記(1)〜(11)のいずれかに記載のリポソームの製造方法。
(15)上記(1)〜(14)のいずれかに記載のリポソームの製造方法で得られたことを特徴とするリポソーム。
(16)熱力学的に安定であることを特徴とする、上記(15)に記載のリポソーム。
(17)上記(15)または(16)に記載のリポソームからなる、生体または細胞に物質を導入するための担体。
(18)生体または細胞に導入される物質が、薬剤、生理活性物質または遺伝子であることを特徴とする、上記(17)記載の担体、
(19)上記(15)または(16)に記載のリポソームを含有し、薬剤、生理活性物質もしくは遺伝子を該リポソームに導入、または担持せしめたことを特徴とする医薬品。
In the general formula 15, R 151 represents a saturated or unsaturated linear or branched aliphatic hydrocarbon group.
(11) The method for producing a liposome according to any one of (1) to (10) above, wherein the glycolipid is a glycolipid biosurfactant compound.
(12) The method for producing a liposome according to any one of the above (1) to (11), wherein the liposome is prepared by changing the chemical structure of a glycolipid constituting the formed coacervate.
(13) The coacervate thus formed is prepared by mixing a phospholipid or a glycolipid other than the glycolipid forming the coacervate, according to any one of (1) to (11) above The manufacturing method of the liposome.
(14) The liposome according to any one of (1) to (11) above, wherein the liposome is prepared by applying electrical stimulation to the formed coacervate or changing pressure or temperature. Manufacturing method.
(15) A liposome obtained by the method for producing a liposome according to any one of (1) to (14) above.
(16) The liposome according to (15) above, which is thermodynamically stable.
(17) A carrier for introducing a substance into a living body or cells, comprising the liposome according to (15) or (16) above.
(18) The carrier according to (17) above, wherein the substance introduced into the living body or cell is a drug, a physiologically active substance or a gene,
(19) A pharmaceutical comprising the liposome according to (15) or (16) above, wherein a drug, a physiologically active substance or a gene is introduced or carried on the liposome.
糖脂質が形成するコアセルベートから、リポソームを形成せしめることによって、リポソームに薬物、生理活性物質、遺伝子などの物質を効率良く封入でき、かつ室温において水溶液状態で長期間安定なリポソームの製造方法およびリポソームを得た。本発明のリポソーム製造方法および該製造方法により得られたリポソームは、生理活性物質、遺伝子などの物質を効率良く封入でき、かつ水溶液状態で長時間安定なリポソームを提供するため、従来問題となっていたリポソームの常温での長期安定性や物質内包効率の低さを解決するものであり、標的細胞へ高い効率で導入できるような物質導入担体を提供できることとなる。 By forming liposomes from coacervates formed by glycolipids, a method for producing liposomes and liposomes capable of efficiently encapsulating substances such as drugs, physiologically active substances and genes in liposomes and stable in aqueous solution at room temperature for a long time Obtained. Since the liposome production method of the present invention and the liposome obtained by the production method can efficiently encapsulate a physiologically active substance, a substance such as a gene, and provide a liposome that is stable for a long time in an aqueous solution, it has been a problem in the past. It solves the long-term stability of liposomes at room temperature and the low substance inclusion efficiency, and can provide a substance introduction carrier that can be introduced into target cells with high efficiency.
本発明に用いられる糖脂質について説明する。
本発明において、糖脂質として、糖脂質系バイオサーファクタントを用いることができる。
バイオサーファクタントとは、生物由来の両親媒性物質であり、界面活性作用を有するものである。例えば、微生物が産生するバイオサーファクタントとしては、糖脂質系のもの、コリノミコール酸(Corynomycolic acid)等の脂肪酸系のもの、エマルサン(Emulsan)、リポサン(Liposan)等のバイオポリマー系のもの、サーファクチン、ビィスコシン等のリポペプタイド系のもの等、種々のものが知られており、これらは通常の界面活性剤に比べ、1)複数の官能基や光学活性を有する点、2)嵩高い構造や複雑な構造を有する点、3)生理活性(抗微生物、抗腫瘍作用など)を有する点、および4)生分解性や安全性が高い点を有することを特徴とする。本発明は、上記バイオサーファクタントのうち、特に糖脂質系のものを用いる点に特徴を有するものである。
本発明において使用する糖脂質としては、例えば、トレハロースリピド系化合物、サクシノイルトレハロースリピド系化合物、ソフォロースリピド系化合物、セロビオースリピド系化合物、マルトースリピド系化合物、ポリオールリピド系化合物、グルコースリピド系化合物、フルクトースリピド系化合物、グルコシドリピド系化合物、マンノシドリピド系化合物、ラムノースリピド系化合物、シュークロースリピド系化合物、アルカノイル−N−グルカミドリピド系化合物等の各種化合物を挙げることができる。また、本発明において、これらの誘導体も糖脂質として使用することができる。
The glycolipid used in the present invention will be described.
In the present invention, a glycolipid biosurfactant can be used as the glycolipid.
A biosurfactant is an amphiphile derived from living organisms and has a surface-active action. For example, biosurfactants produced by microorganisms include glycolipid-based ones, fatty acid-based ones such as corinomycolic acid, biopolymer-based ones such as Emulsan and Liposan, surfactin, Various types such as lipopeptides such as biscosine are known, and these have 1) a plurality of functional groups and optical activity, and 2) a bulky structure and complicated compared to ordinary surfactants. It is characterized by having a structure, 3) a physiological activity (antimicrobial, antitumor action, etc.), and 4) a high biodegradability and safety. The present invention is characterized in that, among the above biosurfactants, particularly glycolipid-based ones are used.
Examples of the glycolipid used in the present invention include trehalose lipid compounds, succinoyl trehalose lipid compounds, sofolose lipid compounds, cellobiose lipid compounds, maltose lipid compounds, polyol lipid compounds, and glucose lipid compounds. And various compounds such as fructose lipid compounds, glucoside lipid compounds, mannoside lipid compounds, rhamnose lipid compounds, sucrose lipid compounds, and alkanoyl-N-glucamide lipid compounds. In the present invention, these derivatives can also be used as glycolipids.
本発明において好ましく用いられる糖脂質としては、上記(2)〜(10)記載の一般式で表された化合物であり、これらで示された一般式の定義中、飽和または不飽和で直鎖または分岐鎖の脂肪族炭化水素基のうち、好ましいものは炭素数1〜36の直鎖または分岐鎖を有するアルキルまたはアルケニル基であり、脂肪族アシル基としては炭素数2〜37のものが好ましい。 The glycolipid preferably used in the present invention is a compound represented by the general formula described in the above (2) to (10), and in the definition of the general formula shown by these, it is saturated or unsaturated, linear or Of the branched aliphatic hydrocarbon groups, preferred are linear or branched alkyl or alkenyl groups having 1 to 36 carbon atoms, and aliphatic acyl groups having 2 to 37 carbon atoms are preferred.
さらに、本発明において特に好ましい糖脂質を以下に例示する。これら化合物を使用する場合、1種のものを単独で用いてもよいし、2種以上のものを混合物の形態で用いることもできる。 Furthermore, particularly preferred glycolipids in the present invention are exemplified below. When these compounds are used, one kind may be used alone, or two or more kinds may be used in the form of a mixture.
まず、本発明に用いられるマンノシドリピド系化合物について説明する。
マンノシドリピド系化合物としては、一般式2で表される化合物が好ましく、一般式2で表される化合物のうち、R25がCH2(OH)−〔CH(OH)〕m2−CH2−基(ただしm2=1〜8、好ましくは2〜6の整数を示す。)であり、R21〜R24が同一もしくは異なっていても良い、炭素数1〜15のアルカノイル基である化合物がより好ましく、一般式16で表されるマンノシルエリスリトール系化合物(以下、MEL−Aとも略す)がさらに好ましい。
First, the mannoside lipid compound used in the present invention will be described.
As the mannoside lipid compound, a compound represented by the general formula 2 is preferable, and among the compounds represented by the general formula 2, R 25 represents a CH 2 (OH) — [CH (OH)] m 2 —CH 2 — group ( However, m2 = 1 to 8, preferably an integer of 2 to 6.), and R 21 to R 24 may be the same or different, and a compound having 1 to 15 carbon atoms is more preferable, A mannosyl erythritol compound represented by the general formula 16 (hereinafter also abbreviated as MEL-A) is more preferable.
(一般式16中、R161およびR162はそれぞれアセチル基(以下、Acとも略す。)を表し、n16は4〜14の整数を表す。) (In General Formula 16, R 161 and R 162 each represent an acetyl group (hereinafter also abbreviated as Ac), and n16 represents an integer of 4 to 14).
また、マンノシドリピド系化合物としては、アルキルマンノシド系の化合物(一般式1において、R18がアルキル基のもの。)も好ましく、その中では一般式17で表される化合物(ML−1)が特に好ましい。 Further, as the mannoside lipid compound, an alkyl mannoside compound (in the general formula 1, R 18 is an alkyl group) is also preferable, and among them, the compound (ML-1) represented by the general formula 17 is Particularly preferred.
(一般式17中、R178はドデシル基を表し、R171、R172、R173およびR174はそれぞれ水素原子を表す。) (In General Formula 17, R 178 represents a dodecyl group, and R 171 , R 172 , R 173, and R 174 each represent a hydrogen atom.)
次に、本発明に用いられるラムノースリピド系化合物について説明する。
ラムノースリピド系化合物の好ましい具体例(RL−1、RL−2、RL−3、RL−4、RL−A、RL−B)を以下に示すが、本発明はこれに限定されるものではない。
Next, the rhamnose lipid compound used in the present invention will be described.
Preferred specific examples (RL-1, RL-2, RL-3, RL-4, RL-A, RL-B) of rhamnose lipid compounds are shown below, but the present invention is not limited thereto. .
次に、本発明に用いられるソフォロースリピド系化合物について説明する。
ソフォロースリピド系化合物の好ましい具体例(SL−1、SL−2、SL−3、SL−5、SL−6)を以下に示すが、本発明はこれに限定されるものではない。
Next, the sofollowus lipid compound used in the present invention will be described.
Preferable specific examples (SL-1, SL-2, SL-3, SL-5, SL-6) of the sofollowus lipid compound are shown below, but the present invention is not limited thereto.
次に、本発明に用いられるトレハロースリピド系化合物について説明する。
トレハロースリピド系化合物の好ましい具体例(TL−1、TL−2)を以下に示すが、本発明はこれに限定されるものではない。
Next, the trehalose lipid compound used in the present invention will be described.
Preferred specific examples (TL-1, TL-2) of trehalose lipid compounds are shown below, but the present invention is not limited thereto.
次に、本発明に用いられるサクシノイルトレハロースリピド系化合物について説明する。
サクシノイルトレハロースリピド系化合物の好ましい具体例(STL−1、STL−2、STL−3)を以下に示すが、本発明はこれに限定されるものではない。
Next, the succinoyl trehalose lipid compound used in the present invention will be described.
Preferred specific examples (STL-1, STL-2, STL-3) of succinoyl trehalose lipid compounds are shown below, but the present invention is not limited thereto.
次に、本発明に用いられるセロビオースリピド系化合物について説明する。
セロビオースリピド系化合物の好ましい具体例(CL−A、CL−B、CL−C)を以下に示すが、本発明はこれに限定されるものではない。
Next, the cellobiose lipid compound used in the present invention will be described.
Preferred specific examples (CL-A, CL-B, CL-C) of cellobiose lipid compounds are shown below, but the present invention is not limited thereto.
次に、本発明に用いられるグルコシドリピド系化合物について説明する。
グルコシドリピド系化合物として、アルキルグルコシド系の化合物(一般式14において、R148がアルキル基のもの。)が好ましい。グルコシドリピド系化合物の好ましい具体例(GL−1)を以下に示すが、本発明はこれに限定されるものではない。
Next, the glucosidolipid compound used in the present invention will be described.
As the glucoside lipid compound, an alkyl glucoside compound (in the general formula 14, R 148 is an alkyl group) is preferable. Although the preferable specific example (GL-1) of a glucoside lipid type compound is shown below, this invention is not limited to this.
(上記式中、R238はドデシル基を表し、R231、R232、R233およびR234はそれぞれ水素原子を表す。) (In the above formula, R 238 represents a dodecyl group, and R 231 , R 232 , R 233 and R 234 each represent a hydrogen atom.)
次に、本発明に用いられるアルカノイル−N−メチルグルカミドリピド系化合物について説明する。
アルカノイル−N−メチルグルカミドリピド系化合物としては、上記一般式15において、R151が炭素数12の飽和または不飽和で、直鎖または分岐鎖の脂肪族炭化水素基である場合が好ましい。
Next, the alkanoyl-N-methylglucamide lipid compound used in the present invention will be described.
As the alkanoyl-N-methylglucamide lipid compound, in the general formula 15, R 151 is preferably a saturated or unsaturated group having 12 carbon atoms and a linear or branched aliphatic hydrocarbon group.
次に、本発明のリポソームの製造方法について説明する。
本発明においては、糖脂質を水中に懸濁してコアセルベートを形成させた後、糖脂質の化学構造を変化させることにより、リポソームを調製する。
Next, the manufacturing method of the liposome of this invention is demonstrated.
In the present invention, a liposome is prepared by suspending a glycolipid in water to form a coacervate and then changing the chemical structure of the glycolipid.
次に、コアセルベートについて説明する。
2種類の高分子水溶液を混合したり、高分子や界面活性剤に塩やアルコールを添加することにより、コロイドに富む液相(濃厚水溶液)と、コロイドに乏しい液相(希薄水溶液)の2液に相分離する(コアセルベーション)。この2相に分離した水溶液のうち、コロイドに富む相をコアセルベートという。
本発明では、糖脂質を用い水中で形成したコアセルベートを経て、リポソームを形成する。コアセルベートは水中に存在する薬剤、タンパク質、酵素、医薬品等の溶質をその内部に多量に保持できる性質がある。従って、有機溶媒を用いて製造するような、従来法により得られるリポソームと比べて、コアセルベートより形成される本発明のリポソームは、より多量に且つ簡便に前記のような溶質を内包できる。
本発明において、コアセルベートの形成法として、例えば、糖脂質等の両性界面活性剤を水中に添加し、攪拌するだけでコアセルベートを形成する方法が挙げられるが、本発明においてはこれに限定されることはない。
コアセルベートを形成するための糖脂質の濃度は特に制限されないが、好ましくは1乃至50mMである。
Next, coacervate will be described.
Two liquids, a liquid phase rich in colloids (concentrated aqueous solution) and a liquid phase poor in colloids (dilute aqueous solution), by mixing two types of aqueous polymer solutions and adding salts and alcohols to polymers and surfactants Phase separation (coacervation). Of the aqueous solution separated into the two phases, the colloid-rich phase is called coacervate.
In the present invention, liposomes are formed through coacervates formed in water using glycolipids. Coacervate has the property of retaining a large amount of solutes such as drugs, proteins, enzymes, and pharmaceuticals present in water. Therefore, the liposome of the present invention formed from coacervate can encapsulate the solute as described above in a larger amount and more easily than liposomes obtained by conventional methods such as those produced using an organic solvent.
In the present invention, a method for forming coacervate includes, for example, a method in which an amphoteric surfactant such as a glycolipid is added to water and stirred to form a coacervate. However, the present invention is limited to this. There is no.
The concentration of glycolipid for forming coacervate is not particularly limited, but is preferably 1 to 50 mM.
上記方法により調製したコアセルベートを含む水溶液に、アセチルエステラーゼなどの酵素を添加し脱アセチル化反応等を引き起こすことにより、もしくは、アルカリ性水溶液を添加し加水分解反応を引き起こすこと等により、糖脂質の化学構造を変化させ、リポソームを調製することができる。
ここで、糖脂質の化学構造の変化とは、糖脂質の1位〜6位の炭素原子に結合する水酸基とのエステル化反応により付加された基(例えば、前記一般式1における、R11〜R18等が挙げられる)に対し、生化学的反応または化学的反応により、加水分解による脱離反応や、別の新たな基を付加する付加反応を行うことによりコアセルベートの形態を変化させて、結果リポソームを形成するような、糖脂質の構造変化をいう。
The chemical structure of glycolipids by adding an enzyme such as acetylesterase to cause deacetylation reaction by adding an enzyme such as acetylesterase to the aqueous solution containing coacervate prepared by the above method, or by causing hydrolysis reaction by adding alkaline aqueous solution Thus, liposomes can be prepared.
Here, the change in the chemical structure of the glycolipid means a group added by an esterification reaction with a hydroxyl group bonded to the carbon atom at the 1st to 6th positions of the glycolipid (for example, R 11 to R 11 in the general formula 1). R 18, etc. to be mentioned are) found by biochemical reaction or chemical reaction, and elimination of the protecting group by hydrolysis, by changing the form of coacervate by performing an addition reaction of adding another new group, As a result, it refers to the structural change of glycolipids to form liposomes.
また、リポソームを調製する別の態様としては、上記方法により、コアセルベートを形成させた後、該コアセルベートを構成する糖脂質とは別の糖脂質、またはリン脂質等の公知の油脂を混合することで、リポソームを得ることができる。
リン脂質またはコアセルベートを構成する糖脂質とは別の糖脂質の添加量としては、コアセルベートを構成する糖脂質に対して、好ましくは10乃至80mol%、さらに好ましくは30mol%である。
本発明に用いられるリン脂質としては、フォスファチジルコリン、ホスファチジルセリン等の公知の中性リン脂質が挙げられ、具体的には、卵ホスファチジルコリン、ジパルミトイルホスファチジルコリンおよびジステアロイルホスファチジルコリン等が挙げられる。
本発明に用いられる、コアセルベートを構成する糖脂質とは別の糖脂質としては、前記糖脂質系バイオサーファクタント等が挙げられる。
As another embodiment for preparing liposomes, coacervate is formed by the above method, and then a known lipid such as a phospholipid or a phospholipid other than the glycolipid constituting the coacervate is mixed. Liposomes can be obtained.
The addition amount of the glycolipid different from the glycolipid constituting the phospholipid or the coacervate is preferably 10 to 80 mol%, more preferably 30 mol% with respect to the glycolipid constituting the coacervate.
Examples of the phospholipid used in the present invention include known neutral phospholipids such as phosphatidylcholine and phosphatidylserine, and specifically, egg phosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine and the like.
Examples of the glycolipid different from the glycolipid constituting the coacervate used in the present invention include the glycolipid biosurfactant.
また、リポソームを調製するさらに別の態様としては、上記方法により、コアセルベートを形成させた後、電気的刺激を与えたり、圧力、温度等を変化させることによりリポソームを調製することができる。具体的には、(1)コアセルベートを含む水溶液に、10乃至200Vのパルス電圧を加えることにより、(2)コアセルベートを含む水溶液の圧力を1barから10bar以上に加圧することにより、もしくは、(3)コアセルベートを含む水溶液の温度を常温(25℃)から50℃以上に加温することにより、リポソームを調製することができる。 Moreover, as another aspect of preparing the liposome, the liposome can be prepared by applying an electrical stimulus or changing the pressure, temperature, etc. after forming the coacervate by the above method. Specifically, (1) by applying a pulse voltage of 10 to 200 V to the aqueous solution containing coacervate, (2) pressurizing the aqueous solution containing coacervate from 1 bar to 10 bar or more, or (3) Liposomes can be prepared by heating the temperature of the aqueous solution containing coacervate from room temperature (25 ° C.) to 50 ° C. or higher.
一般に、糖脂質、りん脂質、界面活性剤等は、水などの媒体中で自ら集合(自己組織化)し、リポソーム、コアセルベート、若しくは、キュービック液晶等、様々な分子集合体を形成する。このうち、リポソームは、その内部に巨大な内水相を有している。
これに対し、MEL−A等の糖脂質は、水中に懸濁すると、リポソームに見られるような内水相は持たず、集合体の内部にも脂質が詰まっている油滴のようなコアセルベートとなる場合がある。このコアセルベートに対して、上記方法を実施することで、コアセルベートからリポソームへと形態が変化する。
In general, glycolipids, phospholipids, surfactants, and the like assemble themselves (self-assembly) in a medium such as water to form various molecular aggregates such as liposomes, coacervates, or cubic liquid crystals. Among these, the liposome has a huge inner aqueous phase.
On the other hand, glycolipids such as MEL-A, when suspended in water, do not have an internal aqueous phase as seen in liposomes, and coacervates such as oil droplets clogged with lipids inside the aggregate. There is a case. By carrying out the above method on this coacervate, the form changes from coacervate to liposome.
上記方法により形成されたリポソームは、常温に3ヶ月以上放置しておいても、安定に分散することができる。すなわち、本発明のリポソームは、熱力学的に安定である。ここで、熱力学的に安定であるとは、リポソームの構造、及び分散状態が安定であることをいう。
本発明で調製されるリポソームは一枚膜でも多重膜のものでも良い。
また、従来の方法により製造されるリポソームは、通常、サイジング処理等を行わなければ、その粒子は数μm以上となる。しかし、本発明によれば、このような処理を行うことなく、100〜1,000nm程度の粒子径を持つリポソームを得ることができる。
Liposomes formed by the above method can be stably dispersed even when left at room temperature for 3 months or more. That is, the liposome of the present invention is thermodynamically stable. Here, being thermodynamically stable means that the structure and dispersion state of the liposome are stable.
Liposomes prepared in the present invention may be monolayer or multilamellar.
Moreover, the liposome manufactured by the conventional method usually has a particle size of several μm or more unless sizing treatment or the like is performed. However, according to the present invention, a liposome having a particle size of about 100 to 1,000 nm can be obtained without performing such treatment.
また、本発明のコアセルベートを形成する原料脂質としては、糖脂質、好ましくは、糖脂質系バイオサーファクタント化合物の1種又は2種類以上を膜成分の全部又は一部として使用するが、所望により、非イオン性界面活性剤を用いることもできる。非イオン性界面活性剤としては、例えば、アルキル又はアルケニルポリオキシアルキレンエーテル、ポリオキシアルキレンアルキル又はアルケニルフェニルエーテル型、脂肪酸ポリオキシアルキレンエステル、アルキロールアマイド型、脂肪酸ポリオキシアルキレンソルビタンエステル、脂肪酸ポリオキシアルキレンソルビトールエステル、ポリオキシアルキレンひまし油、アルキル又はアルケニルポリオキシアルキレンアミン、アルキル又はアルケニルポリオキシアルキレンアミド、又は脂肪酸ソルビタンエステル、脂肪酸ソルビトールエステル、脂肪酸ポリグリセリンエステル、脂肪酸ショ糖エステル等の多価アルコール型及びアルキロールアミド型、ポリエーテル変性シリコーン型界面活性剤、ポリオキシアルキレングリコール型、アルキレングリコール脂肪酸エステル型、ポリアルキレングリコール脂肪酸エステル型、ポリオキシアルキレンソルビット脂肪酸エステル型、ポリオキシアルキレンソルビトール脂肪酸エステル型、グリセリン脂肪酸エステル型又はモノグリセリド有機酸エステル、ポリグリセリンエステル等の誘導体、ポリオキシアルキレングリセリン脂肪酸エステル型、ショ糖脂肪酸エステル型又はその誘導体、ポリオキシアルキレンショ糖脂肪酸エステル型、アミノ酸型、およびこれら2種類以上の混合物を挙げることができる。 In addition, as the raw lipid for forming the coacervate of the present invention, one or more of glycolipids, preferably glycolipid-based biosurfactant compounds are used as all or part of the membrane component. An ionic surfactant can also be used. Nonionic surfactants include, for example, alkyl or alkenyl polyoxyalkylene ether, polyoxyalkylene alkyl or alkenyl phenyl ether type, fatty acid polyoxyalkylene ester, alkylol amide type, fatty acid polyoxyalkylene sorbitan ester, fatty acid polyoxy Polyhydric alcohol types such as alkylene sorbitol ester, polyoxyalkylene castor oil, alkyl or alkenyl polyoxyalkylene amine, alkyl or alkenyl polyoxyalkylene amide, or fatty acid sorbitan ester, fatty acid sorbitol ester, fatty acid polyglycerin ester, fatty acid sucrose ester Alkylolamide type, polyether-modified silicone type surfactant, polyoxyalkylene glycol type, Kirene glycol fatty acid ester type, polyalkylene glycol fatty acid ester type, polyoxyalkylene sorbite fatty acid ester type, polyoxyalkylene sorbitol fatty acid ester type, glycerin fatty acid ester type or monoglyceride organic acid ester, derivatives of polyglycerin ester, polyoxyalkylene glycerin Examples thereof include fatty acid ester types, sucrose fatty acid ester types or derivatives thereof, polyoxyalkylene sucrose fatty acid ester types, amino acid types, and mixtures of two or more of these.
また、コアセルベートに所望の薬剤、生理活性物質または遺伝子等の物質を添加すれば、これらの物質をリポソームの内水相(水性内部)に所望の濃度で封入したリポソームを容易に得ることができる。そして本発明のリポソームは、分散安定性に優れ、かつ薬剤、生理活性物質または遺伝子等の物質の封入効率が高いため、有効な治療等に用いることができる。 Moreover, if a desired drug, physiologically active substance, or gene substance is added to the coacervate, a liposome in which these substances are encapsulated in the inner aqueous phase (aqueous interior) of the liposome at a desired concentration can be easily obtained. The liposome of the present invention is excellent in dispersion stability and has high encapsulation efficiency of substances such as drugs, physiologically active substances or genes, and therefore can be used for effective treatment.
また、本発明のリポソームは、その表面に遺伝子等の物質を結合させて複合体を形成することにより、遺伝子等の物質を担持させることができる。
本発明により得られるリポソームに遺伝子を担持させようとする場合においては、目的とする遺伝子を含む発現ベクターを構築し、上記コアセルベートに該発現ベクター添加、混合し、上記方法によりリポソームを調製せしめれば、該発現ベクターを担持したリポソームを容易に得ることができる。
なお、本発明のリポソームに遺伝子を担持させる場合、該リポソームの構成物質の一部として、陽イオン性脂質が含まれていることが好ましい。陽イオン性脂質としては、コレスレリル−3β−カルボキシアミドエチレン−N−ヒドロキシエチルアミンやコレスレリル−3β−カルボキシアミドエチレン−N,N−ジメチルアミン等が挙げられ、これらの化合物の合成法は、R.Okayamaら、「FEBS Letters」(408、232−234、1997)等に記載されている。
Moreover, the liposome of the present invention can carry a substance such as a gene by binding a substance such as a gene to the surface thereof to form a complex.
In the case of trying to carry a gene in the liposome obtained by the present invention, if an expression vector containing the target gene is constructed, the expression vector is added to the coacervate and mixed, and the liposome is prepared by the above method. A liposome carrying the expression vector can be easily obtained.
In addition, when a gene is carried on the liposome of the present invention, it is preferable that a cationic lipid is contained as a part of the constituent material of the liposome. Examples of cationic lipids include cholesrelyl-3β-carboxyamidoethylene-N-hydroxyethylamine, cholesrelyl-3β-carboxyamidoethylene-N, N-dimethylamine, and the like. Okayama et al., “FEBS Letters” (408, 232-234, 1997) and the like.
上記方法により調製した、薬剤、生理活性物質または遺伝子等の物質をリポソームの水性内部に導入したリポソーム、もしくは、リポソームの表面に担持したリポソームを、水又は生理食塩水等に懸濁して、例えば培養細胞に投与したり、生体内に静脈内注射することができるので、本発明のリポソームは遺伝子治療等において極めて有用なものである。
また、薬剤等を導入、担持させたリポソームを、化粧品に含有させることもできる。
A liposome prepared by the above method, in which a drug, a physiologically active substance, or a gene is introduced into the aqueous interior of the liposome, or a liposome supported on the surface of the liposome is suspended in water or physiological saline, for example, cultured. Since it can be administered to cells or injected intravenously into a living body, the liposome of the present invention is extremely useful in gene therapy and the like.
Moreover, the liposome which introduce | transduced and carry | supported the chemical | medical agent etc. can also be contained in cosmetics.
本発明のリポソームに導入、または、担持される遺伝子としては、特にその種類により限定されるものではないが、生理活性物質等をコードする遺伝子、例えばα−、β−又はγ−インターフェロン遺伝子、G−CSF遺伝子、肝炎ウイルスのアンチセンスをコードする遺伝子等が挙げられる。
本発明のリポソームに導入し得る薬剤および生理活性物質としては、特にその種類により限定されるものではないが、リン酸L−アスコルビルマグネシウム、アスコルビン酸グリコシド、グリチルリチン酸ジカリウム、エスチン、エスクリン、インドメタシン、アスコルビン酸、またはこれらの塩など等が挙げられる。
The gene introduced into or carried by the liposome of the present invention is not particularly limited by the type thereof, but a gene encoding a physiologically active substance, such as an α-, β- or γ-interferon gene, G -CSF gene, gene encoding hepatitis virus antisense, and the like.
The drug and physiologically active substance that can be introduced into the liposome of the present invention are not particularly limited by the kind thereof, but include L-ascorbyl magnesium phosphate, ascorbyl glycoside, dipotassium glycyrrhizinate, estine, esculin, indomethacin, ascorbine. Examples thereof include acids or salts thereof.
また、本発明のリポソーム、特に、糖脂質系バイオサーファクタント化合物からなるリポソームは、構成成分が天然物の由来のものであるため、生体に投与した場合に、従来のリポソームに比較して毒性が低いという利点がある。 In addition, the liposome of the present invention, in particular, a liposome composed of a glycolipid biosurfactant compound is derived from a natural product, and therefore has low toxicity when administered to a living body compared to conventional liposomes. There is an advantage.
以下、本発明の実施例を示すが、本発明は特にこれらにより限定されるものではない Examples of the present invention will be described below, but the present invention is not particularly limited thereto.
(参考例)
一般式16で示すn16=10のものを主成分とするMEL−Aを1〜50mMとなるように試験管中に測りとり、蒸留水2mLを加えて1分間ボルテックス処理したところ、コロイド状の水溶液が得られた。MEL−Aの濃度が2mMのときの水溶液をNikon社製の微分干渉光学顕微鏡であるE600(商品名)を用いて観察した結果を図1に示す。この図から明らかなように、水溶液中にMEL−Aが形成する粒子径2μm〜20μmのコアセルベートが多数観察された(図1中、球状部に相当する)。さらに、MEL−Aの濃度の増加に伴い、コアセルベートの粒子径は増大し、MEL−Aの濃度が50mMのときでは、コアセルベートの粒子径は100μm程度となった。
(Reference example)
MEL-A mainly composed of n16 = 10 represented by the general formula 16 was measured in a test tube so as to be 1 to 50 mM, and 2 mL of distilled water was added and vortexed for 1 minute to obtain a colloidal aqueous solution. was gotten. The result of observing the aqueous solution when the concentration of MEL-A is 2 mM using E600 (trade name) which is a differential interference optical microscope manufactured by Nikon is shown in FIG. As is clear from this figure, many coacervates with a particle diameter of 2 μm to 20 μm formed by MEL-A in the aqueous solution were observed (corresponding to the spherical portion in FIG. 1). Furthermore, as the concentration of MEL-A increased, the particle size of coacervate increased. When the concentration of MEL-A was 50 mM, the particle size of coacervate was about 100 μm.
(実施例1)
参考例で得られた、前記MEL−Aが形成するコアセルベートに、シグマ社製の酵素であるアセチルエステラーゼ1nMを作用させることによって、MEL−Aの糖鎖部分を脱アセチル化した。この際のコアセルベートの形態変化をカールツアイス社製の共焦点レーザー操作顕微鏡を用いて観察した。その結果について、脱アセチル化処理前の共焦点レーザー操作顕微鏡写真を図2−1(a)に、脱アセチル化処理後の共焦点レーザー操作顕微鏡写真を図2−2(a)に示す。また、図2−1(b)および図2−2(b)は、図2−1(a)および図2−2(a)中の矢印に沿って測定した蛍光強度を示すグラフを表し、図2−1(b)および図2−2(b)中、横軸は図2−1(a)および図2−2(a)中の矢印の距離を示し、縦軸は、蛍光強度を示す。これらの図から明らかなように、糖鎖部分を脱アセチル化処理することによって、コアセルベートから、内部に内水相を持つリポソームを調製できた。
Example 1
The glycerin portion of MEL-A was deacetylated by allowing acetylesterase 1 nM, an enzyme manufactured by Sigma, to act on the coacervate formed by MEL-A obtained in Reference Example. At this time, the change in the shape of the coacervate was observed using a confocal laser operating microscope manufactured by Carl Zeiss. Regarding the results, FIG. 2-1 (a) shows a confocal laser operation micrograph before deacetylation, and FIG. 2-2 (a) shows a confocal laser operation micrograph after deacetylation. Moreover, FIGS. 2-1 (b) and 2-2 (b) represent the graph which shows the fluorescence intensity measured along the arrow in FIGS. 2-1 (a) and 2-2 (a), In FIGS. 2-1 (b) and 2-2 (b), the horizontal axis indicates the distance of the arrows in FIGS. 2-1 (a) and 2-2 (a), and the vertical axis indicates the fluorescence intensity. Show. As is clear from these figures, liposomes having an internal aqueous phase inside can be prepared from coacervate by deacetylating the sugar chain portion.
(実施例2)
MEL−Aの代わりに、ラムノースリピド系化合物としてRL−4、ソフォロースリピド系化合物としてSL−5、トレハロースリピド系化合物としてTL−1(m20=14、n20=16のもの)、セロビオースリピド系化合物としてCL−C(R221がOHで、X=4のもの)、グルコシドリピド系化合物としてドデシル−β−D−グルコシド(GL−1)、マンノシド系リピド化合物としてドデシル−β−D−マンノシド(ML−1)、または、アルカノイル−N−メチルグルカミドリピド系化合物としてドデシル−N−メチルグルカミドを用いて、それぞれ参考例と同様の操作を行ったところ、参考例と同様、コアセルベートを調製できた。なお、これらの糖脂質は、北本大、“オレオサイエンス”、1(1)、17−31(2001)記載の方法によって微生物培養液より調製した。また、実施例1と同様に、上記のコアセルベートを形成している糖脂質の脱アセチル化処理を行うことによって、コアセルベートからリポソームが形成された。
(Example 2)
Instead of MEL-A, RL-4 as rhamnose lipid compound, SL-5 as sophorus lipid compound, TL-1 (m20 = 14, n20 = 16) as trehalose lipid compound, cellobiose lipid system CL-C ( R221 is OH, X = 4) as a compound, dodecyl-β-D-glucoside (GL-1) as a glucosidolipid compound, dodecyl-β-D-mannoside (as a mannoside lipid compound) ML-1) or dodecyl-N-methylglucamide as an alkanoyl-N-methylglucamide lipid compound, and the same operations as in the Reference Example were performed. As in the Reference Example, coacervate could be prepared. It was. These glycolipids were prepared from a microorganism culture solution by the method described in Kitamoto Univ., “Oreoscience”, 1 (1), 17-31 (2001). Moreover, the liposome was formed from the coacervate by performing the deacetylation process of the glycolipid which forms said coacervate similarly to Example 1. FIG.
(実施例3)
蛍光物質であるカルセイン(商品名、株式会社同仁研究所社製)0.1mMを含む緩衝液(10mM Tris−HCl(pH7.3))中で、参考例と同様、MEL−Aを用いてコアセルベートを調製した。これに、ジパルミトイルフォスファチジルコリン(DPPC)の添加量が、MEL−Aに対して0mol%〜100mol%となるよう、10mol%刻みで、DPPCを添加し、リポソームを形成した。
次に、この調製したリポソーム懸濁液40μLを、2.0mLの10mM Tris−HCl(pH7.3)緩衝液に添加し、蛍光強度を測定した(励起波長は490nm、蛍光波長は520nmとした)。このときの蛍光強度をFtとする。使用した装置は、蛍光分光光度計FP−6500(商品名、日本電子株式会社)である。
次に、10mMのCoCl2水溶液を20μL添加し、リポソームに封入されなかったカルセインを消光させ、リポソーム内部に封入されたカルセインのみの蛍光強度を測定した。このときの蛍光強度をFinとする。
さらに20%TritonX−100(商品名)溶液を20μL添加することにより、リポソームを破壊し、すべてのカルセインをCo2+と結合させ消光させる。このときの蛍光強度をFqとする。
リポソームの保持効率は、以下の式により計算される。
Example 3
In the buffer solution (10 mM Tris-HCl (pH 7.3)) containing 0.1 mM of calcein (trade name, manufactured by Dojin Laboratories Co., Ltd.), which is a fluorescent substance, coacervate using MEL-A as in the Reference Example. Was prepared. To this, DPPC was added in increments of 10 mol% so that the amount of dipalmitoylphosphatidylcholine (DPPC) added was 0 mol% to 100 mol% with respect to MEL-A to form liposomes.
Next, 40 μL of the prepared liposome suspension was added to 2.0 mL of 10 mM Tris-HCl (pH 7.3) buffer, and fluorescence intensity was measured (excitation wavelength was 490 nm, and fluorescence wavelength was 520 nm). . The fluorescence intensity at this time is Ft. The apparatus used is a fluorescence spectrophotometer FP-6500 (trade name, JEOL Ltd.).
Next, 20 μL of 10 mM CoCl 2 aqueous solution was added to quench the calcein that was not encapsulated in the liposome, and the fluorescence intensity of only calcein encapsulated inside the liposome was measured. The fluorescence intensity at this time is defined as Fin.
Furthermore, by adding 20 μL of a 20% Triton X-100 (trade name) solution, the liposome is broken, and all calcein is bound to Co 2+ and quenched. The fluorescence intensity at this time is defined as Fq.
The retention efficiency of the liposome is calculated by the following formula.
上記式中、rはリポソーム懸濁液及び試薬の添加に伴う反応液の体積変化を補正した値を示し、本実施例においてはr=(2.0mL(Tris緩衝液)+0.04mL(カルセインを含むリポソーム懸濁液)+0.02mL(CoCl2水溶液)+0.02mL(TritonX−100溶液))/2.0mL=1.04である。ここで、保持効率とは、リポソームに保持させたい薬物、生理活性物質のリポソームへの封入効率を示している。その結果を図3に示す。
図3より明らかなように、保持効率を持たないコアセルベートから、10mol%〜80mol%、特に、30mol%のDPPCの添加によって、保持効率の大きなリポソームへと形態変化することが確認できた。
なお、DPPC以外のリン脂質であるジステアロイルフォスファチジルコリン(DSPC)、ジミリストリルフォスファチジルコリン(DMPC)、ジラウロイルフォスファチジルコリン(DLPC)を用いても、同様の結果が得られた。
In the above formula, r represents a value obtained by correcting the volume change of the reaction solution accompanying the addition of the liposome suspension and the reagent. In this example, r = (2.0 mL (Tris buffer solution) +0.04 mL (calcein is added). Liposome suspension) +0.02 mL (CoCl 2 aqueous solution) +0.02 mL (Triton X-100 solution)) / 2.0 mL = 1.04. Here, retention efficiency indicates the encapsulation efficiency of a drug or physiologically active substance to be retained in the liposome. The result is shown in FIG.
As can be seen from FIG. 3, it was confirmed that the coacervate having no retention efficiency changed into a liposome having a large retention efficiency by addition of 10 mol% to 80 mol%, particularly 30 mol% DPPC.
Similar results can be obtained using distearoyl phosphatidylcholine (DSPC), dimyristol phosphatidylcholine (DMPC), or dilauroyl phosphatidylcholine (DLPC) other than DPPC. It was.
(実施例4)
実施例3で形成したリポソームのうち、MEL−Aに対して、DPPCを30mol%添加して調製したリポソームの粒子径を、大塚電子(株)製の光散乱光度計を用いて測定した。その結果を図4に示す。図4からわかるように、本発明のリポソームの粒子径は、約600nmと極めて小さいことが確認できた。
Example 4
Among the liposomes formed in Example 3, the particle diameter of liposomes prepared by adding 30 mol% of DPPC to MEL-A was measured using a light scattering photometer manufactured by Otsuka Electronics Co., Ltd. The result is shown in FIG. As can be seen from FIG. 4, it was confirmed that the particle diameter of the liposome of the present invention was as extremely small as about 600 nm.
(実施例5)
実施例3で調製したリポソームを、常温(25℃)で3ヶ月間静置した後の、リポソームの安定性を検討した。
その結果、実施例3において保持効率が高かった、DPPCをMEL−Aに対して10mol%〜80mol%添加した場合、その他の組成の水溶液に比べて均一な水溶液が得られた。また、この組成においては、得られたリポソームは常温で3ヶ月以上静置していても熱力学的に安定であることがわかった。
(Example 5)
The stability of the liposomes after the liposomes prepared in Example 3 were allowed to stand at room temperature (25 ° C.) for 3 months was examined.
As a result, when DPPC was added in an amount of 10 mol% to 80 mol% with respect to MEL-A, which had high retention efficiency in Example 3, a uniform aqueous solution was obtained as compared with aqueous solutions of other compositions. Moreover, in this composition, it was found that the obtained liposomes were thermodynamically stable even when allowed to stand at room temperature for 3 months or more.
(実施例6)
参考例で形成したMEL−Aのコアセルベートに、蛍光ラベルしたヒト抗体であるシグマ社製のFITC−hIgG(商品名)を20mg/ml添加、攪拌して、カールツアイス社製の共焦点レーザー操作顕微鏡を用いて観察した。
その結果、コアセルベート内部からのみ、FITC−hIgGの蛍光が確認され、コアセルベート内部に前記人抗体を添加したヒト抗体の総量のうち80乃至90%を濃縮して内包させることが可能であった。
なお、FITC−hIgGの代わりに、Promega社製のプラスミドDNApGL3(商品名)を用いた場合も、同様の結果が得られた。
(Example 6)
20 mg / ml of FITC-hIgG (trade name) manufactured by Sigma, which is a fluorescently labeled human antibody, was added to the MEL-A coacervate formed in the Reference Example, stirred, and confocal laser operated microscope manufactured by Carl Zeiss Was observed.
As a result, the fluorescence of FITC-hIgG was confirmed only from the inside of the coacervate, and it was possible to concentrate and encapsulate 80 to 90% of the total amount of the human antibody in which the human antibody was added to the coacervate.
Similar results were obtained when plasmid DNApGL3 (trade name) manufactured by Promega was used instead of FITC-hIgG.
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