JP2007137805A - Transport medium composition for gene or the like comprising polyamidoamine dendron lipid - Google Patents
Transport medium composition for gene or the like comprising polyamidoamine dendron lipid Download PDFInfo
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- JP2007137805A JP2007137805A JP2005332011A JP2005332011A JP2007137805A JP 2007137805 A JP2007137805 A JP 2007137805A JP 2005332011 A JP2005332011 A JP 2005332011A JP 2005332011 A JP2005332011 A JP 2005332011A JP 2007137805 A JP2007137805 A JP 2007137805A
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- Prior art keywords
- gene
- lipid
- methanol
- dendron
- solution
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- 125000002889 tridecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
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Abstract
Description
本発明は、遺伝子、薬物、生理活性物質、生理活性ペプチド類、タンパク質（本明細書を通じて「遺伝子等」という）を、目的とする細胞へ運搬するための運搬媒体と運搬媒体組成物および運搬方法に関する。 The present invention relates to a transport medium, a transport medium composition, and a transport method for transporting genes, drugs, bioactive substances, bioactive peptides, and proteins (referred to as “genes and the like” throughout this specification) to target cells. About.
遺伝子治療は治療すべき細胞に治療用遺伝子を導入し、それを発現させることで治療を行う。細胞中に所定の遺伝子を投入するためのカプセルをベクターという。このベクターはもともと細胞に入り込む性質を持つ、ウィルスの「殻」や脂質の小胞（リポソーム）等が使われる。 Gene therapy is performed by introducing a therapeutic gene into the cell to be treated and expressing it. A capsule for introducing a predetermined gene into a cell is called a vector. This vector originally has the property of entering cells, such as viral “shells” or lipid vesicles (liposomes).
ウィルスベクターは、ウィルスの「殻」を用いたベクターで病原性や毒性を持つ遺伝子（以下「ＤＮＡ」ともいう。）を切り取り、代わりに薬となる遺伝子をはめ込んで作成される。ウィルスベクターは、もともと細胞内に入り込む能力を有しているので、細胞内に遺伝子を持ち込む能力には優れている。しかし、切り取ったとは言え、病原性や毒性、増殖性がないとは言い切れない場合もあり、安全面では課題が残る。 A viral vector is a vector using a “shell” of a virus, which is created by cutting out a gene having pathogenicity or toxicity (hereinafter also referred to as “DNA”) and inserting a gene serving as a drug instead. Since a viral vector originally has the ability to enter cells, it is excellent in the ability to bring genes into cells. However, although it has been cut out, there are cases where it cannot be said that there is no pathogenicity, toxicity, or proliferation, and there are still problems in terms of safety.
非ウィルスベクターは、リポゾームベクターとも呼ばれ、細胞膜に良く似た構造の脂質二重膜でできたミクロの球体で、この中に遺伝子を入れ、使用する。病原性や毒性はないものを作れるが、細胞中に外来の遺伝子を導入して発現させるトランスフェクション効率には課題が残る。すなわち、安全性の観点から非ウィルスベクターのトランスフェクション効率の向上が求められる。 Non-viral vectors, also called liposome vectors, are microspheres made of lipid bilayers with a structure very similar to cell membranes, into which genes are used. Although there are no pathogenicity or toxicity, there is a problem with the transfection efficiency in which foreign genes are introduced and expressed in cells. That is, improvement in transfection efficiency of non-viral vectors is required from the viewpoint of safety.
また、遺伝子はリポソームの中に封入されても、あるいはリポソームと複合体を形成していても細胞内に取り込まれることができる。リポソームとＤＮＡの複合体はリポプレックスと呼ばれる。 Further, the gene can be taken up into the cell even if it is encapsulated in the liposome or forms a complex with the liposome. The complex of liposome and DNA is called a lipoplex.
ベクターに包まれたＤＮＡは、エンドサイトーシスという過程を経て細胞内に取り込まれる。取り込まれたベクターは、エンドソームという細胞小器官に保持され、やがてリソソームによって分解されてしまう。 The DNA wrapped in the vector is taken up into the cell through a process called endocytosis. The taken-in vector is held in an organelle called an endosome and is eventually degraded by lysosomes.
従って、トランスフェクション効率を上げるには、ベクターは分解される前に、エンドソームから脱出し、ＤＮＡを細胞質内にリリースすることが必要である。非ウィルスベクターは、プロトンスポンジ効果やベクターとエンドソームの膜が一体となる膜融合という機能を保有することによって、エンドソームからＤＮＡをリリースする。 Thus, to increase transfection efficiency, the vector must escape from the endosome and release DNA into the cytoplasm before being degraded. Non-viral vectors release DNA from endosomes by possessing a function of proton sponge effect and membrane fusion in which the vector and endosomal membranes are integrated.
本発明者はすでにデンドロンタイプの脂質が、上記のプロトンスポンジ効果と膜融合の機能を有し、効率の高いトランスフェクション効率を有する非ウィルスベクターとなりえる点を示した（特許文献１参照）。 The present inventor has already shown that a dendron type lipid can be a non-viral vector having the above-described proton sponge effect and membrane fusion function and high transfection efficiency (see Patent Document 1).
ベクターを遺伝子治療に用いるにはさらに標的とする細胞に特異的に吸着する能力が求められる。また、ベクター同士はお互いに凝集して大きくなる傾向があるが、一定の大きさより小さい方が投与方法の制限がなくなる点で望ましい。
本発明は上記の課題を解決するために先に示したデンドロンタイプの脂質を改良することで発想されたものである。
In order to use a vector for gene therapy, the ability to specifically adsorb to a target cell is required. In addition, vectors tend to aggregate and become larger with each other, but smaller ones are desirable because there is no restriction on the administration method.
The present invention has been conceived by improving the dendron type lipid described above in order to solve the above-mentioned problems.
デンドロン脂質の極性基が有している多数の機能性分子導入サイトにリガンドを導入する。また、デンドロン脂質にＰＥＧ（ポリエチレングリコール）等の高分子を導入する。 A ligand is introduced into a number of functional molecule introduction sites possessed by polar groups of dendron lipids. Also, a polymer such as PEG (polyethylene glycol) is introduced into the dendron lipid.
デンドロン脂質にリガンドを導入することによって、リガンドが特異的に吸着するレセプターを表面に有する細胞に対して標的指向性のあるベクターを構築することができる。これは遺伝子導入効率が高く、かつ細胞毒性の低い遺伝子運搬媒体であるから、DDS（Drug Delivery System）として用いることができる。また、デンドロン脂質にPEG等の高分子を導入することで、ベクターの大きさが一定以下の大きさになり、イン・ビボ（in vivo）での使用が容易となる。 By introducing a ligand into a dendron lipid, a target-directed vector can be constructed for cells having a receptor on the surface to which the ligand specifically adsorbs. Since this is a gene delivery medium with high gene transfer efficiency and low cytotoxicity, it can be used as a DDS (Drug Delivery System). Further, by introducing a polymer such as PEG into the dendron lipid, the size of the vector becomes a certain size or less, and it is easy to use in vivo.
・基本となるデンドロン脂質
本発明に用いるデンドロン脂質としては、以下に示されるＤＬ−Ｇ１〜ＤＬ−Ｇ８の化合物である。
ＤＬ−Ｇ１：Ｒ1Ｒ2ＮＸ（ＸＨ2）2
ＤＬ−Ｇ２：Ｒ1Ｒ2ＮＸ（Ｘ（ＸＨ2）2）2
ＤＬ−Ｇ３：Ｒ1Ｒ2ＮＸ（Ｘ（Ｘ（ＸＨ2）2）2）2
ＤＬ−Ｇ４：Ｒ1Ｒ2ＮＸ（Ｘ（Ｘ（Ｘ（ＸＨ2）2）2）2）2
ＤＬ−Ｇ５：Ｒ1Ｒ2ＮＸ（Ｘ（Ｘ（Ｘ（Ｘ（ＸＨ2）2）2）2）2）2
ＤＬ−Ｇ６：Ｒ1Ｒ2ＮＸ（Ｘ（Ｘ（Ｘ（Ｘ（Ｘ（ＸＨ2）2）2）2）2）2）2
ＤＬ−Ｇ７：Ｒ1Ｒ2ＮＸ（Ｘ（Ｘ（Ｘ（Ｘ（Ｘ（Ｘ（ＸＨ2）2）2）2）2）2）2）2
ＤＬ−Ｇ８：Ｒ1Ｒ2ＮＸ（Ｘ（Ｘ（Ｘ（Ｘ（Ｘ（Ｘ（Ｘ（ＸＨ2）2）2）2）2）2）2）2）2
式中 Ｒ1及びＲ2は、同一または異なったアルキル基、アルコキシ基、アリール基またはアラルキル基を示す。Ｘは、−ＣＨ2ＣＨ2ＣＯＮＨＣＨ2ＣＨ2Ｎ−を示す。
· The dendrons lipids used in the underlying dendrons lipids present invention is a compound of DL-G1~DL-G8 shown below.
DL-G1: R 1 R 2 NX (XH 2 ) 2
DL-G2: R 1 R 2 NX (X (XH 2 ) 2 ) 2
DL-G3: R 1 R 2 NX (X (X (XH 2 ) 2 ) 2 ) 2
DL-G4: R 1 R 2 NX (X (X (X (XH 2 ) 2 ) 2 ) 2 ) 2
DL-G5: R 1 R 2 NX (X (X (X (X (XH 2 ) 2 ) 2 ) 2 ) 2 ) 2
DL-G6: R 1 R 2 NX (X (X (X (X (X (XH 2) 2) 2) 2) 2) 2) 2
DL-G7: R 1 R 2 NX (X (X (X (X (X (X (XH 2 ) 2 ) 2 ) 2 ) 2 ) 2 ) 2 ) 2 ) 2
DL-G8: R 1 R 2 NX (X (X (X (X (X (X (X (XH 2 ) 2 ) 2 ) 2 ) 2 ) 2 ) 2 ) 2 ) 2
In the formula, R 1 and R 2 represent the same or different alkyl group, alkoxy group, aryl group or aralkyl group. X represents -CH 2 CH 2 CONHCH 2 CH 2 N-.
上記ＤＬ−Ｇ１〜ＤＬ−Ｇ８のいずれかのポリアミドアミンデンドロン脂質において、アルキル基としては、ヘキシル、ヘプチル、オクチル、ノニル、デシル、ウンデシル、ドデシル、トリデシル、テトラデシル、ヘキサデシル、オクタデシル、エイコサデシル、２−エチルヘキシルなどの炭素数６〜２０の直鎖または分枝を有するアルキル基が挙げられる。 In the polyamidoamine dendron lipid of any of the DL-G1 to DL-G8, the alkyl group includes hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, hexadecyl, octadecyl, eicosadecyl, 2-ethylhexyl. And an alkyl group having a straight chain or a branched chain having 6 to 20 carbon atoms.
アルコキシ基としては、ヘキシルオキシ、ヘプチルオキシ、オクチルオキシ、ノニルオキシ、デシルオキシ、ウンデシルオキシ、ドデシルオキシ、トリデシルオキシ、テトラデシルオキシ、ヘキサデシルオキシ、オクタデシルオキシ、エイコシルオキシ、２−エチルヘキシルオキシなどの炭素数６〜２０の直鎖または文枝を有するアルコキシ基が挙げられる。 Examples of the alkoxy group include hexyloxy, heptyloxy, octyloxy, nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy, hexadecyloxy, octadecyloxy, eicosyloxy, 2-ethylhexyloxy, etc. An alkoxy group having a straight chain or a branch having 6 to 20 carbon atoms is exemplified.
アリール基としては、ベンジル、ナフチル、ビフェニル、アントラニル、フェナントリルなどが挙げられる。アラルキル基としては、ベンジル、フェネチルなどが挙げられる。 Aryl groups include benzyl, naphthyl, biphenyl, anthranyl, phenanthryl and the like. Aralkyl groups include benzyl, phenethyl and the like.
Ｘは、−ＣＨ2ＣＨ2ＣＯＮＨＣＨ2ＣＨ2Ｎ−を表し、その末端のＮは、通常２個の水素原子を有するが、1個の水素原子がロイシン、バリン、イソロイシン、ノルロイシン、フェニルアラニン、チロシンなどの疎水性アミノ酸で置換されていてもよい。 X represents —CH 2 CH 2 CONHCH 2 CH 2 N—, and the terminal N usually has two hydrogen atoms, but one hydrogen atom is leucine, valine, isoleucine, norleucine, phenylalanine, tyrosine. And may be substituted with a hydrophobic amino acid.
本発明に用いるポリアミドアミンデンドロン脂質は、例えば以下のようにして製造することができる。 The polyamidoamine dendron lipid used in the present invention can be produced, for example, as follows.
本発明に用いる好ましいポリアミドアミンデンドロン脂質を以下に示す。
・導入するリガンド
標的指向性を持たせるために、ポリアミドアミンデンドロン脂質の機能性分子導入サイトに付加するリガンドとしては、ガラクトース等の糖類を用いる。癌細胞の表面に比較的多く存在するレセプターのリガンドと考えられるからである。しかし、付加するリガンドは標的とする相手側のレセプターに応じて変更することができ、糖類に限定されるものではない。
-Ligand to be introduced As a ligand to be added to the functional molecule introduction site of the polyamidoamine dendron lipid, a saccharide such as galactose is used in order to have target directivity. This is because it is considered to be a ligand of a receptor that is relatively abundant on the surface of cancer cells. However, the ligand to be added can be changed according to the target receptor of the other party, and is not limited to sugars.
・導入するリガンドの割合
これらのリガンドは、ポリアミドアミンデンドロン脂質のすべての導入サイトにあるのが望ましいが、すべての導入サイトになくても本件発明の効果は得ることができる。
-Ratio of ligands to be introduced These ligands are preferably present at all introduction sites of the polyamidoamine dendron lipid, but the effects of the present invention can be obtained even if they are not present at all introduction sites.
・導入方法
基体となるポリアミドアミンデンドロン脂質のアミノ結合の部分が、機能性分子導入サイトとなる。従って、この部分に結合せさる方法であれば、特に限定されるものではない。例としてはリガンドの一部をエステル化したものを上記サイトと結合させたり、リガンドの糖の１部分を酸にして、ポリアミドアミンデンドロン脂質と直接結合させるなどの方法がある。
-Introduction method The amino bond portion of the polyamidoamine dendron lipid serving as the substrate serves as a functional molecule introduction site. Therefore, there is no particular limitation as long as it is a method of bonding to this portion. Examples include a method in which a part of the ligand is esterified and bound to the site, or a part of the sugar of the ligand is acidified and directly bound to the polyamidoamine dendron lipid.
・ＰＥＧの修飾について
他に付加する機能性分子としてはＰＥＧなどの高分子がある。これらをポリアミドデンドロン脂質の表面に付加することで、ベクターの凝集が抑制される。その他、ポリビニルアルコールやポリビニルピロリドンなど水溶性の高分子が使用可能である。
-Modification of PEG Other functional molecules to be added include polymers such as PEG. By adding these to the surface of the polyamide dendron lipid, aggregation of the vector is suppressed. In addition, water-soluble polymers such as polyvinyl alcohol and polyvinyl pyrrolidone can be used.
これらの高分子は末端にイソシアナートやカルボキシル基、スクシミジル基、イミダゾリルカーボネート基を持つように改変した後、デンドロン脂質の末端アミノ基と反応させる。なお、付加する分子や付加方法について、特に限定されるものではない。 These polymers are modified to have an isocyanate, carboxyl group, succimidyl group, or imidazolyl carbonate group at the end, and then reacted with the terminal amino group of the dendron lipid. The molecule to be added and the addition method are not particularly limited.
なお、リガンドまたはＰＥＧ等の高分子を結合させたポリアミドアミンデンドロン脂質を修飾デンドロン脂質と呼ぶ。 A polyamidoamine dendron lipid to which a polymer such as a ligand or PEG is bound is called a modified dendron lipid.
遺伝子等運搬媒体組成物
本発明の遺伝子等運搬媒体組成物は、修飾デンドロン脂質の他、ポリアミドアミンデンドロン脂質やリン脂質を好適に含むことができる。このようなリン脂質としては、ホスファチジルエタノールアミン、ホスファチジルコリン、ホスファチジルセリン、ホスファチジルイノシトール、ホスファチジルグリセロール、カルジオリピン、スフィンゴミエリン、プラスズローゲンおよびホスファチジン酸等を挙げることができ、これらは１種または２種以上を組み合わせて用いることができる。
Gene transportation medium composition The gene transportation medium composition of the present invention can suitably contain a polyamidoamine dendron lipid and a phospholipid in addition to the modified dendron lipid. Examples of such phospholipids include phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin, plaslogen, and phosphatidic acid, and these include one or more. They can be used in combination.
このうち、ホスファチジルエタノールアミンおよびホスファチジルコリンをそれぞれ単独で、または組み合わせて用いるのが好ましい。これらのリン脂質の脂肪酸残基は、特に限定されるべきものではないが、炭素数１２から１８の飽和または不飽和の脂肪酸残基をあげることができ、具体的には、ラウロイル基、ミリストイル基、パルミトイル基、ステアロイル基、オレオイル基、リノレイル基等をあげることができ、ＤＯＰＥ（ジオレオイルホスファチジルエタノールアミン）が特に好ましい。 Of these, phosphatidylethanolamine and phosphatidylcholine are preferably used alone or in combination. The fatty acid residue of these phospholipids is not particularly limited, and examples thereof include saturated or unsaturated fatty acid residues having 12 to 18 carbon atoms, specifically, lauroyl group, myristoyl group. , Palmitoyl group, stearoyl group, oleoyl group, linoleyl group and the like, and DOPE (dioleoylphosphatidylethanolamine) is particularly preferable.
・リン脂質の配合量
リン脂質の配合量は特に限定されないが、リン脂質と修飾デンドロン脂質とポリアミドアミンデンドロン脂質の合計量を１００重量部とした場合にリン脂質３０〜９０重量部、修飾デンドロン脂質とポリアミドアミンデンドロン脂質７０〜１０重量部、好ましくはリン脂質５０〜８０重量部、修飾デンドロン脂質とポリアミドアミンデンドロン脂質５０〜２０重量部、より好ましくはリン脂質６０〜７０重量部、修飾デンドロン脂質とポリアミドアミンデンドロン脂質４０〜３０重量部である。
-Blending amount of phospholipid The blending amount of phospholipid is not particularly limited, but when the total amount of phospholipid, modified dendron lipid and polyamidoamine dendron lipid is 100 parts by weight, phospholipid 30-90 parts by weight, modified dendron lipid And polyamidoamine dendron lipid 70 to 10 parts by weight, preferably phospholipid 50 to 80 parts by weight, modified dendron lipid and polyamidoamine dendron lipid 50 to 20 parts by weight, more preferably 60 to 70 parts by weight phospholipid and modified dendron lipid Polyamide amine dendron lipid is 40 to 30 parts by weight.
・その他の添加剤
リン脂質の他に、遺伝子等運搬媒体組成物に含有され得る添加剤としては、コレステロールなどが例示される。
Other additives In addition to phospholipids, cholesterol and the like are exemplified as an additive that can be contained in the gene transport medium composition.
・デンドロン脂質と遺伝子の配合割合
修飾デンドロン脂質とポリアミドアミンデンドロン脂質を遺伝子等運搬媒体とした場合、遺伝子と遺伝子等運搬媒体の配合割合は、遺伝子１重量部に対して、遺伝子等運搬媒体を１〜２０重量部、好ましくは３〜１５重量部、より好ましくは５〜７重量部使用する。また、遺伝子等運搬媒体とリン脂質の混合物を用いる場合、遺伝子と遺伝子等運搬媒体導の配合割合は、遺伝子１重量部に対し、遺伝子等運搬媒体導を１〜５０重量部、好ましくは５〜３０重量部、より好ましくは１０〜１５重量部使用する。
-Blend ratio of dendron lipid and gene When modified dendron lipid and polyamidoamine dendron lipid are used as a gene transport medium, the ratio of gene and gene transport medium is 1 gene transport medium per 1 part by weight of gene. -20 parts by weight, preferably 3-15 parts by weight, more preferably 5-7 parts by weight. Further, when a mixture of a gene transport medium and a phospholipid is used, the mixing ratio of the gene and the gene transport medium guide is 1 to 50 parts by weight, preferably 5 to 5 parts by weight of the gene transport medium guide to 1 part by weight of the gene. 30 parts by weight, more preferably 10 to 15 parts by weight is used.
・使用する遺伝子
遺伝子としては、オリゴヌクレオチド、ＤＮＡおよびＲＮＡのいずれでもよく、特に形質転換等のイン・ビトロにおける導入用遺伝子や、イン・ビボで発現することにより作用する遺伝子、例えば、遺伝子治療用遺伝子、実験動物や家畜等の産業用動物の品種改良に用いられる遺伝子が好ましい。遺伝子治療用遺伝子としては、アンチセンスオリゴヌクレオチド、アンチセンスＤＮＡ、アンチセンスＲＮＡ、酵素、サイトカイン等の生理活性物質をコードする遺伝子、リポザイム、ｓｉＲＮＡなどを挙げることができる。
-The gene gene to be used may be any of oligonucleotide, DNA and RNA, and in particular a gene for introduction in vitro such as transformation, a gene that acts by expression in vivo, for example, for gene therapy Genes and genes used for breeding industrial animals such as laboratory animals and livestock are preferred. Examples of genes for gene therapy include antisense oligonucleotides, antisense DNAs, antisense RNAs, genes encoding physiologically active substances such as enzymes and cytokines, lipozymes, siRNAs and the like.
・使われる細胞
遺伝子が導入される細胞としては、ヒトなどの動物細胞、植物細胞などの真核細胞、細菌などの原核細胞が例示できる。
Examples of the cell into which the cell gene used is introduced include animal cells such as humans, eukaryotic cells such as plant cells, and prokaryotic cells such as bacteria.
・組成物の形態
本発明の組成物の形態としては、修飾デンドロン脂質のみが存在していてもよく、ポリアミドアミンデンドロン脂質とリン脂質のどちらか若しくは両方をさらに混合物として使用していてもよく、修飾デンドロン脂質とポリアミドアミンデンドロン脂質とリン脂質が組み合わさって脂質膜構造体を形成していてもよい。この脂質膜構造体の存在形態およびその製造方法は特に限定されるべきものではないが、例えば、存在形態としては、乾燥した脂質混合物形態、水系溶媒に分散した形態、さらにこれを乾燥させた形態や凍結させた形態等を挙げることができる。
· The form of the composition in the form present invention compositions, modified dendrons may only lipid is present, may also be used as well as mixtures of either or both of the polyamidoamine dendron and phospholipids, The modified dendron lipid, polyamidoamine dendron lipid, and phospholipid may be combined to form a lipid membrane structure. The existence form of the lipid membrane structure and the production method thereof are not particularly limited. For example, the existence form includes a dried lipid mixture form, a dispersed form in an aqueous solvent, and a dried form thereof. And a frozen form.
・乾燥した混合物
乾燥した脂質混合物は、例えば、使用する脂質成分をいったんクロロホルム等の有機溶媒に溶解させ、次いでエバポレータによる減圧乾固や噴霧乾燥機による噴霧乾燥を行うことで製造することができる。
· Dried mixture dried lipid mixture, for example, can be produced by performing a lipid component used was once dissolved in an organic solvent such as chloroform, followed by spray drying with reduced pressure to dryness and the spray dryer by an evaporator.
・分散した形態
脂質膜構造体が水系溶媒に分散した形態としては、一枚膜リポゾーム、多重層リポソーム、O/W型エマルション。Ｗ／Ｏ／Ｗ型エマルション、球状ミセル、ひも状ミセル、不定型の層状構造物などを挙げることができる。分散した状態の脂質膜構造体の大きさは、特に限定されるべきものではないが、例えば、リポソームやエマルションの場合には、粒子系が５０ｎｍから数μｍであり、球状ミセルの場合、粒子系が５ｎｍから５０ｎｍである。ひも状ミセルや不定型の層状構造物の場合は、その１層あたりの厚みが５ｎｍから１０ｎｍでこれらが層を形成していると考えればよい。
-Dispersed morphologies The lipid membrane structure dispersed in an aqueous solvent includes single membrane liposomes, multilamellar liposomes, and O / W emulsions. Examples thereof include W / O / W type emulsion, spherical micelles, string micelles, and irregular layered structures. The size of the lipid membrane structure in a dispersed state is not particularly limited. For example, in the case of liposomes and emulsions, the particle system is 50 nm to several μm, and in the case of spherical micelles, the particle system Is from 5 nm to 50 nm. In the case of a string-like micelle or an irregular layered structure, it can be considered that the thickness per layer is 5 nm to 10 nm and these form a layer.
・溶媒
水系溶媒（分散媒）の組成も特に限定されるべきものではないが、水のほかに、グルコース、乳糖、ショ糖などの糖水溶液、グリセリン、プロピレングリコールなどの多価アルコール水溶液、リン酸緩衝液、クエン酸緩衝液、リン酸緩衝化生理食塩液等の緩衝液、生理食塩水、細胞培養用の培地などを挙げることができる。この水系溶媒に分散した脂質膜構造体を安定に長期間保存するには、凝集などの物理的安定性の面から、水系溶媒中の電解質を極力なくすことが重要である。
-The composition of the solvent aqueous solvent (dispersion medium) is not particularly limited, but besides water, sugar aqueous solutions such as glucose, lactose and sucrose, polyhydric alcohol aqueous solutions such as glycerin and propylene glycol, phosphoric acid Examples of the buffer solution include a buffer solution such as a buffer solution, a citrate buffer solution, and a phosphate buffered physiological saline solution, a physiological saline solution, and a medium for cell culture. In order to stably store the lipid membrane structure dispersed in the aqueous solvent for a long period of time, it is important to eliminate the electrolyte in the aqueous solvent as much as possible from the viewpoint of physical stability such as aggregation.
また、脂質の化学的安定性の面から、水系溶媒中の電解質を極力なくすことが重要である。また、脂質の化学的安定性の面から、水系溶媒のｐＨを弱酸性から中性付近（ｐＨ３．０から８．０）に設定したり、窒素バブリングにより溶存酸素を除去することが重要である。さらに凍結乾燥保存や噴霧乾燥保存をする場合には、糖水溶液を、凍結保存する場合には、糖水溶液や多価アルコール水溶液をそれぞれ用いると効果的な保存が可能である。 From the viewpoint of chemical stability of lipids, it is important to eliminate electrolytes in aqueous solvents as much as possible. From the viewpoint of chemical stability of the lipid, it is important to set the pH of the aqueous solvent from weakly acidic to neutral (pH 3.0 to 8.0) or to remove dissolved oxygen by nitrogen bubbling. . Further, when lyophilized storage or spray-dried storage is used, effective storage is possible by using a sugar aqueous solution, and when storing frozen, a sugar aqueous solution or a polyhydric alcohol aqueous solution is used.
・濃度
これらの水系溶媒の添加物の濃度は特に限定されるべきものではないが、例えば、糖水溶液においては、２から２０％（Ｗ／Ｖ）が好ましく、５から１０％（Ｗ／Ｖ）がさらに好ましい。また、多価アルコール水溶液においては、１から５％（Ｗ／Ｖ）が好ましく、２から２．５％（Ｗ／Ｖ）がさらに好ましい。緩衝液においては、緩衝剤の濃度が１から１５０ｍＭが好ましく、１０から５０ｍＭがさらに好ましい。
Concentration The concentration of these aqueous solvent additives is not particularly limited. For example, in an aqueous sugar solution, 2 to 20% (W / V) is preferable, and 5 to 10% (W / V). Is more preferable. Moreover, in a polyhydric alcohol aqueous solution, 1 to 5% (W / V) is preferable, and 2 to 2.5% (W / V) is more preferable. In the buffer solution, the concentration of the buffer is preferably 1 to 150 mM, and more preferably 10 to 50 mM.
・脂質膜構造体の濃度
水系溶媒中の脂質膜構造体の濃度は、特に限定されるべきものではないが、本発明においては脂質膜構造体として用いるリン脂質の総量の濃度は、０．００１ｍＭから１００ｍＭが好ましく、０．０１ｍＭから２０ｍＭがさらに好ましい。
- concentration of the lipid membrane structure concentration aqueous solvent of the lipid membrane structure, but it should not be particularly limited, the concentration of the total amount of phospholipid used as a lipid membrane structure in the present invention, 0.001 mM To 100 mM, preferably 0.01 mM to 20 mM.
・脂質膜構造体の水に分散したときの形態
脂質膜構造体が水系溶媒に分散した形態は、上記の乾燥した脂質混合物を水系溶媒に添加し、さらにホモジナイザー等の乳化機、超音波乳化機、高圧噴射乳化機等により乳化することで製造することができる。また、リポソームを製造する方法としてよく知られた方法、例えば逆相蒸発法などによっても製造することもでき、特に限定されるべきものではない。脂質膜構造体の大きさを制御したい場合には、孔径のそろったメンブランフィルター等を用いて、高圧下でイクストルージョン（押し出し濾過）を行えばよい。
-Form of lipid membrane structure when dispersed in water The form of lipid membrane structure dispersed in an aqueous solvent is the addition of the above-mentioned dried lipid mixture to an aqueous solvent, and further an emulsifier such as a homogenizer, an ultrasonic emulsifier It can be produced by emulsification with a high-pressure jet emulsifier or the like. Moreover, it can also manufacture by the method well-known as a method of manufacturing a liposome, for example, a reverse phase evaporation method etc., It should not be specifically limited. When it is desired to control the size of the lipid membrane structure, extrusion (extrusion filtration) may be performed under high pressure using a membrane filter having a uniform pore size.
・水系溶媒に分散した脂質膜構造体をさらに乾燥させる
また、上記の水系溶媒に分散した脂質膜構造体をさらに乾燥させる方法としては、通常の凍結乾燥や噴霧乾燥を挙げることができる。このときの水系溶媒としては、上記したように、糖水溶液、好ましくはショ糖水溶液、乳糖水溶液を用いるとよい。ここで、水系溶媒に分散した脂質膜構造体をいったん製造した上でさらに乾燥すると、脂質膜構造体の長期保存が可能となるほか、この乾燥した脂質膜構造体に遺伝子水溶液を添加すると、効率よく脂質混合物が水和されるために遺伝子自身も効率よく、脂質膜構造体に保持されることができるといったメリットがある。
-Further drying the lipid membrane structure dispersed in an aqueous solvent. Examples of the method for further drying the lipid membrane structure dispersed in the aqueous solvent include ordinary freeze drying and spray drying. As the aqueous solvent at this time, as described above, a sugar aqueous solution, preferably a sucrose aqueous solution or a lactose aqueous solution may be used. Here, once a lipid membrane structure dispersed in an aqueous solvent is manufactured and further dried, the lipid membrane structure can be stored for a long period of time, and when a gene aqueous solution is added to the dried lipid membrane structure, the efficiency is increased. Since the lipid mixture is often hydrated, there is an advantage that the gene itself can be efficiently held in the lipid membrane structure.
・その他
本発明の遺伝子等運搬媒体組成物は、遺伝子だけでなく、親水性の大きい薬物、高分子量の生理活性ペプチド類、蛋白質などの細胞内に導入されにくい薬物、生理活性物質、生理活性ペプチド、蛋白質などにも適用できる。本発明の組成物を用いれば、イン・ビトロ及びイン・ビボのいずれにおいても細胞内に遺伝子等を効率良く導入、送達することができる。
And other genes such as delivery vehicle compositions of the present invention not only genes, hydrophilic high drug, physiologically active peptides of high molecular weight, hard drugs are introduced into cells such as proteins, physiologically active substances, physiologically active peptide It can also be applied to proteins. By using the composition of the present invention, genes and the like can be efficiently introduced and delivered into cells both in vitro and in vivo.
イン・ビトロでの遺伝子等導入は、標的とする細胞を含む懸濁液に本発明の遺伝子等運搬媒体組成物を添加したり、本発明の遺伝子等運搬媒体組成物を含有する培地で標的とする細胞を培養するなどの手段により、行うことができる。 In vitro gene introduction may be performed by adding the gene delivery medium composition of the present invention to a suspension containing target cells or by using a medium containing the gene delivery medium composition of the present invention. This can be done by culturing cells to be cultured.
イン・ビボでの遺伝子等の導入は、本発明の含有組成物を宿主に投与すればよい。宿主への投与手段としては、経口投与でもよく、非経口投与でもよいが、非経口投与が好ましい。剤形としては、通常知られたものでよく、経口投与の剤形としては、例えば、錠剤、散剤、顆粒剤、シロップ剤などを上げることができる。 For introduction of a gene or the like in vivo, the containing composition of the present invention may be administered to a host. The means for administration to the host may be oral administration or parenteral administration, but parenteral administration is preferred. The dosage form may be a conventionally known dosage form, and examples of the dosage form for oral administration include tablets, powders, granules, syrups and the like.
また、非経口投与の剤形としては、例えば注射剤、点眼剤、軟膏剤、坐剤等を挙げることができる。中でも注射剤が好ましく、投与方法としては、静脈注射、標的とする細胞や臓器に対しての局所注射が好ましい。 Examples of parenteral dosage forms include injections, eye drops, ointments, suppositories, and the like. Among these, injections are preferable, and as an administration method, intravenous injection and local injection into a target cell or organ are preferable.
実施例１
本実施例では、ＤＬ−Ｇ３にラクトビオン酸を結合させることで、ＤＬ−Ｇ３の末端をガラクトースで修飾する。
Example 1
In this example, the end of DL-G3 is modified with galactose by binding lactobionic acid to DL-G3.
１．ＤＬ−Ｇ３の合成
本実施例に用いるＤＬ−Ｇ３は以下の手順で作成する。なお反応式は化１を参照のこと。
１−１．ＤＬ−Ｇ―０．５の合成
蒸留したアクリル酸メチル（１４ｍｌ、０．１５６ｍｏｌ）にジーｎ−ドデシルアミン（２．００ｇ、５．６６ｍｍｏｌ）を溶かし、窒素雰囲気において８０℃で１８時間還流した。その後、未反応のアクリル酸メチルを減圧留去し、シリカゲル（展開溶媒は、石油エーテル：ジエチルエーテル＝２：１）で精製した。基本となるデンドロン脂質のＲ１Ｒ２がジドデシルタイプのデンドロン脂質であるので、「−２Ｃ１２」と付記する
1. Synthesis of DL-G3 DL-G3 used in this example is prepared by the following procedure. For the reaction formula, see
1-1. Synthesis of DL-G-0.5 Di-n-dodecylamine (2.00 g, 5.66 mmol) was dissolved in distilled methyl acrylate (14 ml, 0.156 mol) and refluxed at 80 ° C. for 18 hours in a nitrogen atmosphere. Thereafter, unreacted methyl acrylate was distilled off under reduced pressure, and purification was performed on silica gel (developing solvent was petroleum ether: diethyl ether = 2: 1). Since R1R2 of the basic dendron lipid is a didodecyl-type dendron lipid, it is added as “−2C12”.
１−２．ＤＬ−Ｇ０−２Ｃ１２の合成
ＤＬ−Ｇ−０．５−２Ｃ１２（２．１６２ｇ、４．９２ｍｍｏｌ）をメタノール（５０ｍｌ、１．２３ｍｏｌ）に溶かした。この溶液を、シアン化ナトリウム（０．０４８ｇ、０．９７９ｍｍｏｌ）を含む蒸留したエチレンジアミン（１００ｍｌ、１．５０ｍｏｌ）に徐徐（以下「徐々」と記す。）に加え、窒素雰囲気において４５℃で５０時間攪拌した。その後、メタノールと未反応のエチレンジアミンを減圧留去し、Ｓｅｐｈａｄａｘ ＬＨ−２０カラム（溶離液としてメタノール）によって精製した。
1-2. Synthesis of DL-G0-2C12 DL-G-0.5-2C12 (2.162 g, 4.92 mmol) was dissolved in methanol (50 ml, 1.23 mol). This solution was added to distilled ethylenediamine (100 ml, 1.50 mol) containing sodium cyanide (0.048 g, 0.979 mmol) gradually (hereinafter referred to as “gradually”), and then in a nitrogen atmosphere at 45 ° C. for 50 hours. Stir. Thereafter, methanol and unreacted ethylenediamine were distilled off under reduced pressure, and the residue was purified by a Sephax LH-20 column (methanol as an eluent).
１−３．ＤＬ−Ｇ０．５−２Ｃ１２の合成
ＤＬ−Ｇ０−２Ｃ１２（２．２５６ｇ。４．８２ｍｍｏｌ）をメタノール（１７．５ｍｌ、０．４３１ｍｏｌ）に溶かした。この溶液を、蒸留したアクリル酸メチル（４３．５ｍｌ。０．４３１ｍｏｌ）に徐々に加え、窒素雰囲気において３５℃で５０時間攪拌した。その後メタノールと未反応のアクリル酸メチルを減圧留去し、シリカゲル（展開溶液として石油エーテル：ジエチルエーテル＝２：１ のち ジクロロメタン：メタノール＝９：１）で精製した。
1-3. Synthesis of DL-G0.5-2C12 DL-G0-2C12 (2.256 g. 4.82 mmol) was dissolved in methanol (17.5 ml, 0.431 mol). This solution was gradually added to distilled methyl acrylate (43.5 ml, 0.431 mol) and stirred at 35 ° C. for 50 hours in a nitrogen atmosphere. Thereafter, methanol and unreacted methyl acrylate were distilled off under reduced pressure, and the residue was purified on silica gel (petroleum ether: diethyl ether = 2: 1 as a developing solution and then dichloromethane: methanol = 9: 1).
１―４．ＤＬ−Ｇ１−２Ｃ１２の合成
ＤＬ−Ｇ０．５−２Ｃ１２（０．７１４ｇ、１．１２ｍｍｏｌ）をメタノール（２０．５ｍｌ、０．５０５ｍｏｌ）に溶かした。この溶液を、シアン化ナトリウム（０．０１１ｇ、０．２２４ｍｍｏｌ）を含む蒸留したエチレンジアミン（３７．５ｍｌ、０．５６２ｍｏｌ）に徐々に加え、窒素雰囲気において４５℃で５０時間攪拌した。その後、メタノールと未反応のエチレンジアミンを減圧留去し、Ｓｅｐｈａｄａｘ ＬＨ−２０カラム（溶離液としてメタノール）によって精製した。
1-4. Synthesis of DL-G1-2C12 DL-G0.5-2C12 (0.714 g, 1.12 mmol) was dissolved in methanol (20.5 ml, 0.505 mol). This solution was gradually added to distilled ethylenediamine (37.5 ml, 0.562 mol) containing sodium cyanide (0.011 g, 0.224 mmol) and stirred at 45 ° C. for 50 hours in a nitrogen atmosphere. Thereafter, methanol and unreacted ethylenediamine were distilled off under reduced pressure, and the residue was purified by a Sephax LH-20 column (methanol as an eluent).
１―５．ＤＬ−Ｇ１．５−２Ｃ１２の合成
ＤＬ−Ｇ１−２Ｃ１２（１．５３９ｇ。２．２１ｍｍｏｌ）をメタノール（８６．５ｍｌ、２．１３ｍｏｌ）に溶かした。この溶液を、蒸留したアクリル酸メチル（１５２ｍｌ。１．６９ｍｏｌ）に徐々に加え、窒素雰囲気において３５℃で５０時間攪拌した。その後メタノールと未反応のアクリル酸メチルを減圧留去し、シリカゲル（展開溶液としてジクロロメタン：メタノール＝９：１）で精製した。
1-5. Synthesis of DL-G1.5-2C12 DL-G1-2C12 (1.539 g. 2.21 mmol) was dissolved in methanol (86.5 ml, 2.13 mol). This solution was gradually added to distilled methyl acrylate (152 ml, 1.69 mol) and stirred at 35 ° C. for 50 hours in a nitrogen atmosphere. Thereafter, methanol and unreacted methyl acrylate were distilled off under reduced pressure, and the residue was purified by silica gel (dichloromethane: methanol = 9: 1 as a developing solution).
１−６．ＤＬ−Ｇ２−２Ｃ１２の合成
ＤＬ−Ｇ１．５−２Ｃ１２（１．７０３ｇ、１．６４ｍｍｏｌ）をメタノール（４６ｍｌ、１．１３ｍｏｌ）に溶かした。この溶液を、シアン化ナトリウム（０．０３２ｇ、０．６５５ｍｍｏｌ）を含む蒸留したエチレンジアミン（１３４ｍｌ、２．００ｍｏｌ）に徐々に加え、窒素雰囲気において４５℃で５０時間攪拌した。その後、メタノールと未反応のエチレンジアミンを減圧留去し、Ｓｅｐｈａｄａｘ ＬＨ−２０カラム（溶離液としてメタノール）によって精製した。
1-6. Synthesis of DL-G2-2C12 DL-G1.5-2C12 (1.703 g, 1.64 mmol) was dissolved in methanol (46 ml, 1.13 mol). This solution was gradually added to distilled ethylenediamine (134 ml, 2.00 mol) containing sodium cyanide (0.032 g, 0.655 mmol) and stirred at 45 ° C. for 50 hours in a nitrogen atmosphere. Thereafter, methanol and unreacted ethylenediamine were distilled off under reduced pressure, and the residue was purified by a Sephax LH-20 column (methanol as an eluent).
１−７．ＤＬ−Ｇ２．５−２Ｃ１２の合成
ＤＬ−Ｇ２−２Ｃ１２（１．７１３ｇ。１．４９ｍｍｏｌ）をメタノール（１２０ｍｌ、２．９６ｍｏｌ）に溶かした。この溶液を、蒸留したアクリル酸メチル（１００ｍｌ。１．１２ｍｏｌ）に徐々に加え、３０℃で２５時間攪拌した。その後メタノールと未反応のアクリル酸メチルを減圧留去し、シリカゲル（展開溶液としてジクロロメタン：メタノール＝８５：１５）で精製した。
1-7. Synthesis of DL-G2.5-2C12 DL-G2-2C12 (1.713 g. 1.49 mmol) was dissolved in methanol (120 ml, 2.96 mol). This solution was gradually added to distilled methyl acrylate (100 ml, 1.12 mol) and stirred at 30 ° C. for 25 hours. Thereafter, methanol and unreacted methyl acrylate were distilled off under reduced pressure, and the residue was purified by silica gel (dichloromethane: methanol = 85: 15 as a developing solution).
１−８．ＤＬ−Ｇ３−２Ｃ１２の合成
ＤＬ−Ｇ２．５−２Ｃ１２（０．１１５ｇ、０．０６３ｍｍｏｌ）をメタノール（２．５３ｍｌ、０．６２５ｍｏｌ）に溶かした。この溶液を、シアン化ナトリウム（０．００１２ｇ、０．０２５ｍｍｏｌ）を含む蒸留したエチレンジアミン（５．００ｍｌ、０．０７５ｍｏｌ）に徐々に加え、窒素雰囲気において４５℃で５０時間攪拌した。その後、メタノールと未反応のエチレンジアミンを減圧留去し、Ｓｅｐｈａｄａｘ ＬＨ−２０カラム（溶離液としてメタノール）によって精製した。以上の工程を繰り返して所定量のＤＬ−Ｇ３−２Ｃ１２を得た。
1-8. Synthesis of DL-G3-2C12 DL-G2.5-2C12 (0.115 g, 0.063 mmol) was dissolved in methanol (2.53 ml, 0.625 mol). This solution was gradually added to distilled ethylenediamine (5.00 ml, 0.075 mol) containing sodium cyanide (0.0012 g, 0.025 mmol) and stirred at 45 ° C. for 50 hours in a nitrogen atmosphere. Thereafter, methanol and unreacted ethylenediamine were distilled off under reduced pressure, and the residue was purified by a Sephax LH-20 column (methanol as an eluent). The above process was repeated to obtain a predetermined amount of DL-G3-2C12.
２．DL-G3−２Ｃ１２とラクトビオン酸とからGal-DL-G3-2C12の合成
Ｎ−ｈｙｄｒｏｘｙｓｕｃｃｉｎｉｍｉｄｅ（０．２４ｇ、２．０９ｍｍｏｌ）とジシクロヘキシカルボジイミド（ＤＣＣ）０．４７ｇ（２．２６ｍｍｏｌ）を、ジメチルホルムアミド（ＤＦＭ）５ｍｌに溶かしたＬａｃｔｏｂｉｏｎｉｃ ａｃｉｄ ラクトビオン酸（０．６２ｇ、１．７３ｍｍｏｌ）に加え、氷冷下で５時間攪拌しＡ液とした。
ＤＬ−Ｇ３−２Ｃ１２（０．２８ｇ、０．１３７ｍｍｏｌ）とトリエチルアミン（０．３６２ｍｌ、２．６１ｍｍｏｌ）をジメチルスルホキシド（ＤＭＳＯ）５ｍｌに溶かし、これをＡ液に加えて室温で７日間攪拌した。その後反応液をろ過し、ろ液をエーテルで２回再沈殿させ、沈殿物を透析（Ｍｗ ｃｕｔｏｆｆ １０００）により精製した（３日間）。
以下に反応式を示す。
2. Synthesis of Gal-DL-G3-2C12 from DL-G3-2C12 and lactobionic acid N-hydroxysuccinimide (0.24 g, 2.09 mmol) and dicyclohexylcarbodiimide (DCC) 0.47 g (2.26 mmol) The solution was added to lactobionic acid lactobionic acid (0.62 g, 1.73 mmol) dissolved in 5 ml of formamide (DFM) and stirred for 5 hours under ice-cooling to give solution A.
DL-G3-2C12 (0.28 g, 0.137 mmol) and triethylamine (0.362 ml, 2.61 mmol) were dissolved in 5 ml of dimethyl sulfoxide (DMSO), and this was added to solution A and stirred at room temperature for 7 days. Thereafter, the reaction solution was filtered, the filtrate was reprecipitated twice with ether, and the precipitate was purified by dialysis (Mw cutoff 1000) (3 days).
The reaction formula is shown below.
図１および図２には、採取物のＮＭＲ分析結果を示す。図１は、（Ａ）ラクトビオン酸、（Ｂ）ＤＬ−Ｇ３−２Ｃ１２単体、（Ｃ）上記採取物のプロトンＮＭＲ（以下「1ＨＮＭＲ」と記す。）の結果を示す。またそれぞれのピーク位置を示す構造も記した。ＮＭＲのチャート上のアラビア数字は、構造式の水素番号と対応している。周波数は４００ＭＨｚである。 1 and 2 show the NMR analysis results of the collected material. FIG. 1 shows the results of proton NMR (hereinafter referred to as “ 1 HNMR”) of (A) lactobionic acid, (B) DL-G3-2C12 alone, and (C) the collected material. A structure indicating each peak position is also shown. The Arabic numerals on the NMR chart correspond to the hydrogen numbers in the structural formula. The frequency is 400 MHz.
図１を参照して、領域IIの部分を（Ｂ）ＤＬ−Ｇ３−２Ｃ１２単体と（Ｃ）上記採取物
で比較するとアミノ基に結合している６番目の水素が低磁場側へシフトしているのがわかる。この領域IIの部分の積分値を（Ｂ）と（Ｃ）で比較すると、（Ｃ）の方が減少してい
た。この減少分は６番目の水素のシフトを考慮した全末端が置換された場合の理論値と非常によい一致を見た。この結果から、デンドロン脂質の全末端のプロトンが糖で修飾されたことがわかる。
Referring to FIG. 1, when comparing the region II with (B) DL-G3-2C12 alone and (C) the above sample, the 6th hydrogen bonded to the amino group is shifted to the low magnetic field side. I can see that Comparing the integrated value of the region II with (B) and (C), (C) was decreased. This decrease was in good agreement with the theoretical value when all ends were replaced considering the 6th hydrogen shift. This result shows that the protons at all terminals of the dendron lipid were modified with sugar.
図２は同じ採取物の13ＣのＮＭＲ（以下「13ＣＮＭＲ」と記す。）の結果を示す。（Ａ）、(Ｂ)、(Ｃ)と構造およびチャート上のアラビア数字については図１と同じである。図２ではさらに２２番および２３番目のカーボン部分の拡大図も同時に示した。図２より、２２番および２３番目のカーボンが高磁場側へシフトしており、このことも全末端が糖で修飾されていることを示している。以上の結果より全末端にガラクトースを修飾したデンドロン脂質を合成したと確認した。 FIG. 2 shows the results of 13 C NMR (hereinafter referred to as “ 13 CNMR”) of the same sample. (A), (B), (C), the structure, and the Arabic numerals on the chart are the same as in FIG. FIG. 2 also shows enlarged views of the 22nd and 23rd carbon parts. From FIG. 2, the 22nd and 23rd carbons are shifted to the high magnetic field side, which also indicates that all ends are modified with sugar. From the above results, it was confirmed that a dendron lipid modified with galactose at all ends was synthesized.
なお図１および図２のデータを示す。括弧中の番号はそれぞれの図に示した構造の番号に対応する。
1HNMR (DMSO): δ 0.85 (1), δ 1.23 and 1.42 (alkyl protons), δ 2.20-2.67 (dendron protons), δ 3.11-5.20 (2-6 and protons of galactosyl residue), δ 7.81 and 7.92 (protons of dendron amide and lactobionamide);
13CNMR (DMSO): δ 14.0 (1), δ 22.1, 26.5, 28.8, 29.0, 29.1 and 31.3 (2), δ 33.1 (5, 10, 15 and 20), δ 36.4 and 36.9 (7, 12 and 17), δ 38.3 and 38.4 (22 and 23), δ 49.5 (4, 9, 14 and 19), δ 52.2 and 52.6 (8, 13 and 18), δ 60.7 (35), δ 62.3 (29), δ 68.3-75.7 (25, 26, 28, 31-34), δ 104.6 (30), δ 171.4, 171.7 and 172.7 (6, 11, 16, 21 and 24).
The data of FIGS. 1 and 2 are shown. The numbers in parentheses correspond to the structure numbers shown in each figure.
1 HNMR (DMSO): δ 0.85 (1), δ 1.23 and 1.42 (alkyl protons), δ 2.20-2.67 (dendron protons), δ 3.11-5.20 (2-6 and protons of galactosyl residue), δ 7.81 and 7.92 ( protons of dendron amide and lactobionamide);
13 CNMR (DMSO): δ 14.0 (1), δ 22.1, 26.5, 28.8, 29.0, 29.1 and 31.3 (2), δ 33.1 (5, 10, 15 and 20), δ 36.4 and 36.9 (7, 12 and 17 ), δ 38.3 and 38.4 (22 and 23), δ 49.5 (4, 9, 14 and 19), δ 52.2 and 52.6 (8, 13 and 18), δ 60.7 (35), δ 62.3 (29), δ 68.3 -75.7 (25, 26, 28, 31-34), δ 104.6 (30), δ 171.4, 171.7 and 172.7 (6, 11, 16, 21 and 24).
３．リポプレックスの調製
上記のＧａｌ−ＤＬ−Ｇ３−２Ｃ１２のＤＭＦ溶液からロータリーエバポレーターを用いて溶媒を除去して、一晩真空乾燥した。その後、この薄膜にＤＬ−Ｇ３−２Ｃ１２のクロロホルム溶液（６６．６μｌ）とＤＯＰＥのクロロホルム溶液（２４０μｌ）を加え、ロータリーエバポレーターを用いて溶媒を除去して、脂質薄膜を形成させた。これにＰＢＳ（０．５ｍｌ）を加え、バス型超音波照射装置を用いて超音波を２分間照射し、脂質分散液を調製した。この分散液を数種々の脂質濃度に調製し、次に、２０ｍＭＴｒｉｓ−ＨＣｌ溶液のプラスミドＤＮＡ溶液（１μｇ／５０μｌ）をこれらの脂質分散液に加えて混合し、室温で３０分インキュベートして種々のＮ／Ｐ比のＧａｌ−ＤＬ−Ｇ３−２Ｃ１２リポプレックスを調整した。なお、ここでＮ／Ｐ比は、ＤＮＡと脂質膜との比率である。
3. Preparation of Lipoplex The solvent was removed from the above-mentioned Gal-DL-G3-2C12 DMF solution using a rotary evaporator, followed by vacuum drying overnight. Thereafter, a DL-G3-2C12 chloroform solution (66.6 μl) and DOPE chloroform solution (240 μl) were added to the thin film, and the solvent was removed using a rotary evaporator to form a lipid thin film. PBS (0.5 ml) was added thereto, and an ultrasonic wave was irradiated for 2 minutes using a bath-type ultrasonic irradiation device to prepare a lipid dispersion. This dispersion is prepared to several different lipid concentrations, and then a 20 mM Tris-HCl solution of plasmid DNA (1 μg / 50 μl) is added to these lipid dispersions, mixed and incubated at room temperature for 30 minutes. An N / P ratio of Gal-DL-G3-2C12 lipoplex was prepared. Here, the N / P ratio is the ratio of DNA to lipid membrane.
４．遺伝子導入
ヒト肝ガン由来のＨｅｐＧ２細胞を２４穴ディッシュ１穴当たり５．０ｘ１０4個になるように撒き、１０％ＦＣＳ（ウシ胎児血清）含有ＤＭＥＭ（ダルコット改変イーグル培地）０．５ｍｌ中、３７℃で２日間培養した。
その後、０．３６ｍＭＣａＣｌ2と０．４２ｍＭＭｇＣｌ2を含むＰＢＳ（ＰＢＳ（＋））で２回洗浄した後、ＦＣＳ非含有ＤＭＥＭ１ｍｌを加え、１穴当たり１μｇのプラスミドＤＮＡを含むリポプレックスを細胞に加えたのち４時間インキュベーションした。その後ＰＢＳ（＋）で３回洗浄して細胞に取り込まれていないリポプレックスを除去し、１０％ＦＣＳ含有ＤＭＥＭ１ｍｌを加えた。４０時間培養後、ルシフェラーゼアッセイにて遺伝子導入活性を評価した。
4). Gene transfer HepG2 cells derived from human liver cancer were seeded at 5.0 × 10 4 per well of a 24-well dish, and 37 ° C. in 0.5 ml of DMEM (Dalcott Modified Eagle Medium) containing 10% FCS (fetal calf serum). For 2 days.
Then, after washing twice with PBS (PBS (+)) containing 0.36 mM CaCl 2 and 0.42 mM MgCl 2 , 1 ml of FCS-free DMEM was added, and a lipoplex containing 1 μg of plasmid DNA per well was added to the cells. After that, it was incubated for 4 hours. Thereafter, the cells were washed 3 times with PBS (+) to remove lipoplexes not taken up by the cells, and 1 ml of DMEM containing 10% FCS was added. After culturing for 40 hours, gene transfer activity was evaluated by luciferase assay.
５．ルシフェラーゼアッセイによる遺伝子導入の評価
遺伝子導入した４０時間後、ＰＢＳ（＋）で３回洗浄し、さらにＰＢＳで１回洗浄した後、細胞を１穴当たり５０μｌの細胞溶解剤に溶かし、エッペンドルフチューブに回収して１２０００ｒｐｍで２分間遠心分離し、その上澄みを回収した。まず、発光基質液を用いて、ルシフェラーゼ活性の定量を行った。ルシフェラーゼ活性の定量は細胞溶解液２０μｌを１３．５μｇのルシフェリンを含んだ発光基質液９５μｌ、アルブミン溶液（５ｍｇ／ｍｌ）５μｌと混合し、ルミノメーターＬｕｍａｔ ＬＢ９５０７（ベルトールドジャパン）を使って２０秒間の発光量を測定した。次に細胞溶解液５μｌｘ３を用いて、細胞内のタンパク質の定量をＣｏｏｍａｓｓｉｅ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｒｅａｇｅｎｔを用いて行った。定量は細胞溶解液５μｌに対してＣｏｏｍａｓｓｉｅ Ｒｅａｇｅｎｔを２５０μｌずつ加え、波長５９５ｎｍの吸光度をプレートリーダーで測定することによってタンパク質濃度を求めた。そして、細胞のタンパク質当たりのルシフェラーゼ活性を求めた。図３にその結果を示す。
5. Evaluation of gene transfer by luciferase assay 40 hours after gene transfer, wash 3 times with PBS (+), 1 time with PBS, then dissolve cells in 50 μl per well of cell lysate and collect in Eppendorf tubes Then, the mixture was centrifuged at 12000 rpm for 2 minutes, and the supernatant was recovered. First, luciferase activity was quantified using a luminescent substrate solution. The luciferase activity was determined by mixing 20 μl of cell lysate with 95 μl of luminescent substrate solution containing 13.5 μg of luciferin and 5 μl of albumin solution (5 mg / ml), and using a luminometer Lumat LB9507 (Berthold Japan) for 20 seconds. The amount of luminescence was measured. Next, the intracellular protein was quantified using Coomassie Protein Assay Reagent using 5 μl × 3 of the cell lysate. Quantification was performed by adding 250 μl of Coomassie Reagent to 5 μl of cell lysate and measuring the absorbance at a wavelength of 595 nm with a plate reader to determine the protein concentration. Then, the luciferase activity per cell protein was determined. The result is shown in FIG.
図３は、リガンド（本実施例の場合はガラクトース）を修飾したデンドロン脂質である、Ｇａｌ−ＤＬ−Ｇ３−２Ｃ１２と修飾していないデンドロン脂質であるＤＬ−Ｇ３−２Ｃ１２と溶媒のＤＯＰＥの混合比をＸ／１／１０とし、Ｎ／Ｐ比（脂質膜とＤＮＡ（遺伝子）の比）毎にＸの値を変えながら、遺伝子導入活性を測定した結果である。横軸はＮ／Ｐ比を、縦軸は遺伝子導入活性を示す。 FIG. 3 shows the mixing ratio of Gal-DL-G3-2C12, which is a dendron lipid modified with a ligand (galactose in this example), and DL-G3-2C12, which is an unmodified dendron lipid, and DOPE as a solvent. Is the result of measuring the gene transfer activity while changing the value of X for each N / P ratio (ratio of lipid membrane to DNA (gene)). The horizontal axis represents the N / P ratio, and the vertical axis represents gene transfer activity.
遺伝子導入活性は、Ｎ／Ｐ比が２の時に最大値を有した。Ｎ／Ｐ比をそれ以上にすると、未修飾のリポプレックスよりも遺伝子活性が低下した。また、Ｎ／Ｐ比を２より小さくすると、遺伝子導入活性の絶対値は低下した。 The gene transfer activity had a maximum value when the N / P ratio was 2. Increasing the N / P ratio reduced gene activity compared to unmodified lipoplexes. In addition, when the N / P ratio was smaller than 2, the absolute value of gene transfer activity was reduced.
しかし、Ｎ／Ｐ比が２の場合は、Ｇａｌ−ＤＬ−Ｇ３−２Ｃ１２を０．１含んでいる場合と含んでいない場合の比率が２：１程度であるのに対して、Ｎ／Ｐ比が１の場合は１０：１程度になっている。
すなわち、Ｇａｌ−ＤＬ−Ｇ３−２Ｃ１２／ＤＬ−Ｇ３−２Ｃ１２／ＤＯＰＥの混合比が０．１／１／１０の時の遺伝子導入の効率は、Ｇａｌ−ＤＬ−Ｇ３−２Ｃ１２を含んでいない場合より高くなった。
However, when the N / P ratio is 2, the ratio when the Gal-DL-G3-2C12 is included and when it is not included is about 2: 1, whereas the N / P ratio is about 2: 1. When 1 is 1, it is about 10: 1.
That is, the efficiency of gene introduction when the mixing ratio of Gal-DL-G3-2C12 / DL-G3-2C12 / DOPE is 0.1 / 1/10 is higher than that when Gal-DL-G3-2C12 is not included. It became high.
これは、Ｎ／Ｐ比が小さくなることで、リポプレックスの静電相互作用による非特異的な取り込みが抑えられ、リガンドの存在による細胞吸着の効果の違いが表れたためと考えられる。すなわち、Ｎ／Ｐ比が１という静電相互作用のない状態では、リガンドのついていないリポプレックスの細胞吸着力は低く、一方リガンドのついているデンドロン脂質を含むリポプレックスは細胞に特異的に吸着することができる。 This is thought to be because nonspecific uptake due to the electrostatic interaction of the lipoplex was suppressed and the difference in the effect of cell adsorption due to the presence of the ligand appeared because the N / P ratio was small. That is, in the state where the N / P ratio is 1 and there is no electrostatic interaction, lipoplexes without ligands have low cell adsorption power, while lipoplexes with dendron lipids with ligands specifically adsorb to cells. be able to.
なおここでは、リポプレックスを調製し遺伝子を細胞に導入することについて実施例を示したが、リポソームを作成し、比較的小さな薬物や生理活性物質を細胞へ送達し、また細胞内に導入することもできる。例えば、具体的なリポソームの調製方法は、以下のようである。 In addition, although the Example was shown about preparing a lipoplex and introducing a gene into a cell here, creating a liposome, delivering a comparatively small drug and physiologically active substance to a cell, and introducing into a cell You can also. For example, a specific method for preparing liposomes is as follows.
卵黄レシチンのクロロホルム溶液（10mg/ml）５００μlをＧａｌ−ＤＬ−Ｇ０−２Ｃ１２のクロロホルム溶液（0.5mg/ml）１０３μlと２０６μlにそれぞれ加えた。また同じく卵黄レシチンのクロロホルム溶液（10mg/ml）５００μlをGal-DL-G3-2C12のDMF溶液（2mg/ml）２９．９μl、７５．２μl、１３７．３μlにそれぞれ加えた。これら５種類の溶液のそれぞれをロータリーエバポレーターで溶媒を除去し薄膜を形成させ、2時間真空乾燥させた。その後、63mMカルセイン水溶液 (pH7.4）500μlを加え、2分間超音波照射し、凍結融解を5回繰り返した後、エクストルーダーを用いて孔径100nmのフィルターを通すことによってリポソ―ム分散液を調製した。さらにセファロース4Bカラムに通すことによってリポソ―ムを精製した。このようにしてデンドロン脂質の異なる５種類のリポソームを実際に作成することができた。
500 μl of a yolk lecithin chloroform solution (10 mg / ml) was added to 103 μl and 206 μl of a Gal-DL-G0-2C12 chloroform solution (0.5 mg / ml), respectively. Similarly, 500 μl of egg yolk lecithin in chloroform (10 mg / ml) was added to 29.9 μl, 75.2 μl, and 137.3 μl of DMF solutions of Gal-DL-G3-2C12 (2 mg / ml), respectively. Each of these five types of solutions was subjected to removal of the solvent with a rotary evaporator to form a thin film, and vacuum dried for 2 hours. Then, add 500 μl of 63 mM calcein aqueous solution (pH 7.4), irradiate with ultrasonic waves for 2 minutes, repeat freeze-
実施例２
ラクトビオン酸（４ｇ、１．１８Ｘ１０-2ｍｏｌ）をメタノールに溶解し、５０℃にてメタノールの減圧留去を４回繰り返し、ラクトビオンラクトンを得た。これをＤＭＳＯ５ｍｌに溶解し、４ｍｌのＤＭＳＯに溶解したＤＬ−Ｇ０−２Ｃ１２（０．４５ｇ、９．５３Ｘ１０-4ｍｏｌ）に滴下して加え、４０℃で１０時間反応させた。その後、透析（Ｍｗ ｃｕｔｏｆｆ２０００）を２日間行い精製した。さらに、ＬＨ−２０により精製した。次にシリカゲルクロマトグラフィー（展開溶媒：クロロホルム／メタノール／水＝６０／３５／５）による精製を行った。そして、透析（Ｍｗ ｃｕｔｏｆｆ２０００）を２日間行い、再度ＬＨ−２０により精製することによりＧａｌ−ＤＬ−Ｇ０−２Ｃ１２を得た。以下に反応式を示す。
Example 2
Lactobionic acid (4 g, 1.18 × 10 −2 mol) was dissolved in methanol, and methanol was distilled off under reduced pressure four times at 50 ° C. to obtain lactobion lactone. This was dissolved in 5 ml of DMSO, added dropwise to DL-G0-2C12 (0.45 g, 9.53 × 10 −4 mol) dissolved in 4 ml of DMSO, and reacted at 40 ° C. for 10 hours. Thereafter, dialysis (Mw cutoff 2000) was performed for 2 days for purification. Furthermore, it refine | purified by LH-20. Next, purification by silica gel chromatography (developing solvent: chloroform / methanol / water = 60/35/5) was performed. Then, dialysis (Mw cutoff 2000) was performed for 2 days, and Gal-DL-G0-2C12 was obtained by purification with LH-20 again. The reaction formula is shown below.
図４には、上記で得たＧａｌ−ＤＬ−Ｇ０−２Ｃ１２の1ＨＮＭＲの結果と構造を示す。Ｇａｌ−ＤＬ−Ｇ０−２Ｃ１２とＤＬ−Ｇ０−２Ｃ１２のピークの違い、特に４．２〜４．６ｐｐｍ付近のシグナルの有無からガラクトース修飾ができており、積分値を理論値と比較することで、ほぼ１００％のＤＬ−Ｇ０が修飾されたことがわかる。図５は、同じく13ＣＮＭＲの結果であるが、やはり７と８のシグナルの変化より、糖での修飾がされていることを示している。このようにして作成したデンドロン脂質も実施例１のデンドロン脂質の代わりに利用することができる。図４および図５のデータを示す。括弧中の番号はそれぞれの図に示した構造の番号に対応する。 FIG. 4 shows the 1 HNMR result and structure of Gal-DL-G0-2C12 obtained above. Galactose modification has been made from the difference in peak between Gal-DL-G0-2C12 and DL-G0-2C12, particularly the presence or absence of a signal in the vicinity of 4.2 to 4.6 ppm. By comparing the integral value with the theoretical value, It can be seen that almost 100% of DL-G0 was modified. FIG. 5 also shows the result of 13 CNMR, but also shows that the modification with sugar is made based on the change in the signals of 7 and 8. The dendron lipid thus prepared can also be used in place of the dendron lipid of Example 1. The data of FIG. 4 and FIG. 5 are shown. The numbers in parentheses correspond to the structure numbers shown in each figure.
1HNMR (CD3OD): δ 0.90 (1), δ 1.30 (2), δ 1.49 (3), δ 2.37 (6), δ 2.49 (4), δ 2.81 (5), δ 3.30-3.94 (7-19);
13CNMR (CD3OD): δ 14.5 (1), δ 23.8, 27.4, 28.6, 30.5, 30.7, 30.8, 31.0 and 33.1 (2), δ 33.7 (5), δ 39.6 and 39.9 (7 and 8), δ 51.0 (4), δ 54.6 (3), δ 62.7 (20), δ 63.8 (14), δ 70.4-77.2 (10, 11, 13, 16-19), δ 83.3 (12), δ 105.7 (15), δ 175.2 (6), δ 175.5 (9).
1 HNMR (CD 3 OD): δ 0.90 (1), δ 1.30 (2), δ 1.49 (3), δ 2.37 (6), δ 2.49 (4), δ 2.81 (5), δ 3.30-3.94 (7 -19);
13 CNMR (CD 3 OD): δ 14.5 (1), δ 23.8, 27.4, 28.6, 30.5, 30.7, 30.8, 31.0 and 33.1 (2), δ 33.7 (5), δ 39.6 and 39.9 (7 and 8), δ 51.0 (4), δ 54.6 (3), δ 62.7 (20), δ 63.8 (14), δ 70.4-77.2 (10, 11, 13, 16-19), δ 83.3 (12), δ 105.7 (15 ), δ 175.2 (6), δ 175.5 (9).
なお、実施例１および２ではラクトビオン酸を用いて修飾デンドロンの末端がガラクトース残基で修飾されるようにしたが、五員環構造若しくは六員環構造を持つ糖類であれば、ラクトビオン酸以外でも容易に置き換えることができる。 In Examples 1 and 2, the end of the modified dendron is modified with a galactose residue using lactobionic acid. However, any sugar other than lactobionic acid can be used as long as it is a saccharide having a five-membered ring structure or a six-membered ring structure. Can be easily replaced.
実施例３
１．DL-G2-2C18とM-PEG５５０からPEG５５０−DL-G2-2C18を合成する。
リポプレックスのｉｎ ｖｉｖｏにおける使用には、血清タンパク質との相互作用が重要な問題である。これは、正に帯電したリポプレックスと負に帯電した血清タンパク質の静電相互作用が原因である。このことによってリポプレックスが崩壊したり、凝集が起こる。発明者がすでに開示したDL-G3-DOPE（特開２００４−１５９５０４号）は、血清中での遺伝子導入が可能である。
Example 3
1. PEG550-DL-G2-2C18 is synthesized from DL-G2-2C18 and M-PEG550.
Interaction with serum proteins is an important issue for the use of lipoplexes in vivo. This is due to electrostatic interactions between positively charged lipoplexes and negatively charged serum proteins. This causes the lipoplexes to collapse or aggregate. DL-G3-DOPE (Japanese Patent Application Laid-Open No. 2004-159504) already disclosed by the inventor is capable of gene transfer in serum.
しかし、その粒径は約８００ｎｍとｉｎ vivoで使用するには若干大きく、表面は正電荷で覆われている。そこで、ポリエチレングリコール（PEG）を結合させた脂質をリポプレックスに導入することによって、リポプレックスを安定化させた。このPEG脂質は主にPEの末端にPEGを結合させたPEG結合デンドロン脂質を合成することで得る。これにより、リポプレックスの安定化およびさらなる血清耐性の向上が期待できる。 However, its particle size is about 800 nm, which is slightly large for use in vivo, and the surface is covered with a positive charge. Therefore, the lipoplex was stabilized by introducing a lipid conjugated with polyethylene glycol (PEG) into the lipoplex. This PEG lipid is obtained mainly by synthesizing a PEG-conjugated dendron lipid in which PEG is bound to the end of PE. Thereby, stabilization of lipoplexes and further improvement of serum resistance can be expected.
実施例１および２では、ジドデシルジアミンを用いてデンドロン脂質のアルキル鎖の部分は炭素１２個のジドデシルタイプのデンドロン脂質を用いたが、ここでは炭素が１８個のジオクタデシルタイプのデンドロン脂質を用いる。以後このタイプのデンドロン脂質に「−２Ｃ１８」をつけて呼ぶ。 In Examples 1 and 2, didodecyldiamine was used and the alkyl chain part of the dendron lipid was a 12-carbon didodecyl-type dendron lipid. Here, a dioctadecyl-type dendron lipid having 18 carbons was used. Use. Hereinafter, this type of dendron lipid is referred to as “-2C18”.
本実施例に用いるＤＬ−Ｇ３−２Ｃ１８は以下の手順で作成する。なお反応式は実施例１，２と同じく化１に従う。
１−１．ＤＬ−Ｇ―０．５−２Ｃ１８の合成
蒸留したアクリル酸メチル（３５ｍｌ、０．３９ｍｏｌ）にジオクタデシルアミン（２．００ｇ、３．８ｍｍｏｌ）を溶かし、窒素雰囲気において８０℃で１８時間還流した。その後、メタノールと未反応のアクリル酸メチルを減圧留去し、未反応のアクリル酸メチルを減圧留去し、シリカゲル（展開溶媒は、石油エーテル：ジエチルエーテル＝２：１）で精製した。
DL-G3-2C18 used for a present Example is produced in the following procedures. The reaction formula follows the
1-1. Synthesis of DL-G-0.5-2C18 Dioctadecylamine (2.00 g, 3.8 mmol) was dissolved in distilled methyl acrylate (35 ml, 0.39 mol) and refluxed at 80 ° C. for 18 hours in a nitrogen atmosphere. Thereafter, methanol and unreacted methyl acrylate were distilled off under reduced pressure, and unreacted methyl acrylate was distilled off under reduced pressure, and purified on silica gel (developing solvent was petroleum ether: diethyl ether = 2: 1).
１−２．ＤＬ−Ｇ０−２Ｃ１８の合成
ＤＬ−Ｇ−０．５（２．０ｇ、３．４ｍｍｏｌ）をメタノール（４０ｍｌ）に溶かした。この溶液を、シアン化ナトリウム（３３ｍｇ、０．６７ｍｍｏｌ）を含む蒸留したエチレンジアミン（７０ｍｌ、１．０５ｍｏｌ）に徐々に加え、窒素雰囲気において５０℃で７日間攪拌した。その後、メタノールと未反応のアクリル酸メチルを減圧留去し、シリカ（展開溶媒：クロロホルム／メタノール／水＝６０／３５／５）により精製した。
1-2. Synthesis of DL-G0-2C18 DL-G-0.5 (2.0 g, 3.4 mmol) was dissolved in methanol (40 ml). This solution was gradually added to distilled ethylenediamine (70 ml, 1.05 mol) containing sodium cyanide (33 mg, 0.67 mmol) and stirred at 50 ° C. for 7 days in a nitrogen atmosphere. Thereafter, methanol and unreacted methyl acrylate were distilled off under reduced pressure, and purification was performed with silica (developing solvent: chloroform / methanol / water = 60/35/5).
１−３．ＤＬ−Ｇ０．５−２Ｃ１８の合成
ＤＬ−Ｇ０（１．２ｇ、１．９ｍｍｏｌ）をメタノール（２０ｍｌ）に溶かした。この溶液を、アクリル酸メチル（３４ｍｌ、０．３８ｍｏｌ）に徐々に加え、窒素雰囲気において３５℃で５０時間攪拌した。その後メタノールと未反応のアクリル酸メチルを減圧留去し、シリカゲル（展開溶液として石油エーテル：ジエチルエーテル＝２：１ のち ジクロロメタン：メタノール＝９：１）で精製した。
1-3. Synthesis of DL-G0.5-2C18 DL-G0 (1.2 g, 1.9 mmol) was dissolved in methanol (20 ml). This solution was gradually added to methyl acrylate (34 ml, 0.38 mol) and stirred at 35 ° C. for 50 hours in a nitrogen atmosphere. Thereafter, methanol and unreacted methyl acrylate were distilled off under reduced pressure, and the residue was purified on silica gel (petroleum ether: diethyl ether = 2: 1 as a developing solution and then dichloromethane: methanol = 9: 1).
１―４．ＤＬ−Ｇ１−２Ｃ１８の合成
ＤＬ−Ｇ０．５（１．５ｇ、１．８ｍｍｏｌ）をメタノール（５０ｍｌ）に溶かした。この溶液を、シアン化ナトリウム（１８ｍｇ、０．３７ｍｍｏｌ）を含む蒸留したエチレンジアミン（６５ｍｌ、０．９２ｍｏｌ）に徐々に加え、窒素雰囲気において４５℃で６０時間攪拌した。その後、メタノールと未反応のエチレンジアミンを減圧留去し、Ｓｅｐｈａｄａｘ ＬＨ−２０カラム（溶離液としてクロロホルム）によって精製した。
1-4. Synthesis of DL-G1-2C18 DL-G0.5 (1.5 g, 1.8 mmol) was dissolved in methanol (50 ml). This solution was gradually added to distilled ethylenediamine (65 ml, 0.92 mol) containing sodium cyanide (18 mg, 0.37 mmol) and stirred at 45 ° C. for 60 hours in a nitrogen atmosphere. Thereafter, methanol and unreacted ethylenediamine were distilled off under reduced pressure, and the residue was purified by a Sephax LH-20 column (chloroform as an eluent).
１―５．ＤＬ−Ｇ１．５−２Ｃ１８の合成
ＤＬ−Ｇ１（１．３ｇ、１．５ｍｍｏｌ）をメタノール（６０ｍｌ）に溶かした。この溶液を、蒸留したアクリル酸メチル（１１０ｍｌ、１．２３ｍｏｌ）に徐々に加え、窒素雰囲気において３５℃で５２時間攪拌した。その後メタノールと未反応のアクリル酸メチルを減圧留去し、シリカゲル（展開溶液としてクロロホルム、後にクロロホルム：メタノール＝９：１）で精製した。
1-5. Synthesis of DL-G1.5-2C18 DL-G1 (1.3 g, 1.5 mmol) was dissolved in methanol (60 ml). This solution was gradually added to distilled methyl acrylate (110 ml, 1.23 mol) and stirred at 35 ° C. for 52 hours in a nitrogen atmosphere. Thereafter, methanol and unreacted methyl acrylate were distilled off under reduced pressure, and the residue was purified by silica gel (chloroform as a developing solution, and later chloroform: methanol = 9: 1).
１−６．ＤＬ−Ｇ２−２Ｃ１８の合成
ＤＬ−Ｇ１．５（１．３ｇ、１．１ｍｍｏｌ）をメタノール（３０ｍｌ）に溶かした。この溶液を、シアン化ナトリウム（２１ｍｇ、０．４３ｍｍｏｌ）を含む蒸留したエチレンジアミン（９５ｍｌ、１．４２ｍｏｌ）に徐々に加え、窒素雰囲気において４５℃で５０時間攪拌した。その後、メタノールと未反応のエチレンジアミンを減圧留去し、Ｓｅｐｈａｄａｘ ＬＨ−２０カラム（溶離液としてメタノール）によって精製した。
1-6. Synthesis of DL-G2-2C18 DL-G1.5 (1.3 g, 1.1 mmol) was dissolved in methanol (30 ml). This solution was gradually added to distilled ethylenediamine (95 ml, 1.42 mol) containing sodium cyanide (21 mg, 0.43 mmol) and stirred at 45 ° C. for 50 hours in a nitrogen atmosphere. Thereafter, methanol and unreacted ethylenediamine were distilled off under reduced pressure, and the residue was purified by a Sephax LH-20 column (methanol as an eluent).
１−７．ＤＬ−Ｇ２．５−２Ｃ１８の合成
ＤＬ−Ｇ２（０．９４ｇ、０．７１ｍｍｏｌ）をメタノール（６０ｍｌ）に溶かした。この溶液を、蒸留したアクリル酸メチル（５５ｍｌ、０．６１ｍｏｌ）に徐々に加え、窒素雰囲気中、３０℃で５０時間攪拌した。その後、メタノールと未反応のアクリル酸メチルを減圧留去し、シリカゲル（展開溶液としてクロロホルム：メタノール＝９５：５のち、クロロホルム：メタノール＝８０：２０）で精製した。
1-7. Synthesis of DL-G2.5-2C18 DL-G2 (0.94 g, 0.71 mmol) was dissolved in methanol (60 ml). This solution was gradually added to distilled methyl acrylate (55 ml, 0.61 mol) and stirred at 30 ° C. for 50 hours in a nitrogen atmosphere. Thereafter, methanol and unreacted methyl acrylate were distilled off under reduced pressure, and the residue was purified by silica gel (chloroform: methanol = 95: 5, then chloroform: methanol = 80: 20 as a developing solution).
１−８．ＤＬ−Ｇ３−２Ｃ１８の合成
ＤＬ−Ｇ２．５（０．９２ｇ、０．４６ｍｍｏｌ）をメタノール（４０ｍｌ）に溶かした。この溶液を、シアン化ナトリウム（８．９ｍｇ、０．１８ｍｍｏｌ）を含む蒸留したエチレンジアミン（６０ｍｌ、０．９ｍｏｌ）に徐々に加え、窒素雰囲気において４５℃で５５時間攪拌した。その後、メタノールと未反応のエチレンジアミンを減圧留去し、Ｓｅｐｈａｄａｘ ＬＨ−２０カラム（溶離液としてメタノール）によって精製した。以上の工程を繰り返して所定量のＤＬ−Ｇ３−２Ｃ１８を得た。
1-8. Synthesis of DL-G3-2C18 DL-G2.5 (0.92 g, 0.46 mmol) was dissolved in methanol (40 ml). This solution was gradually added to distilled ethylenediamine (60 ml, 0.9 mol) containing sodium cyanide (8.9 mg, 0.18 mmol) and stirred at 45 ° C. for 55 hours in a nitrogen atmosphere. Thereafter, methanol and unreacted ethylenediamine were distilled off under reduced pressure, and the residue was purified by a Sephax LH-20 column (methanol as an eluent). The above process was repeated to obtain a predetermined amount of DL-G3-2C18.
Ｍ−ＰＥＧ550４−Ｎｉｔｒｏｐｈｅｎｙｌｃａｒｂｏｎａｔｅ（ＮＰＣ）の合成
ポリエチレングリコール（平均分子量５５０）の一方の末端をメチル基と置換したＭ−ＰＥＧ550を用意する。M-PEG550を０．５ｇ（０．９１ｍｍｏｌ）とトリエチルアミン０．２５２ｍｌ（１．８２ｍｍｏｌ）をテトラヒドロフタン（ＴＨＦ）１０ｍｌに溶解し、これにニトロフェニルクロロホルメイト（４−ｎｉｔｏｒｏｐｈｅｎｙｌｃｈｒｏｌｏｆｏｒｍａｔｅ）０．３６７ｇ（１．８２ｍｍｏｌ）のＴＨＦ溶液（１５ｍｌ）を氷冷下で加え２時間攪拌した。その後、室温で２２時間攪拌した。得られた反応液をろ過し、ろ液を減圧留去後、ＬＨ−２０（溶離液：メタノール）により精製した。これにより得た物質をＭ−ＰＥＧ550−ＮＰＣと記す。以下に反応式を示す。
One end of the synthetic polyethylene glycol (average molecular weight 550) of M-PEG 550 4-Nitrophenylcarbonate ( NPC) providing a M-PEG 550 were replaced with methyl groups. 0.5 g (0.91 mmol) of M-PEG 550 and 0.252 ml (1.82 mmol) of triethylamine were dissolved in 10 ml of tetrahydrophthalane (THF), and 0.367 g of 4-nitrophenylchloroformate was added thereto. A THF solution (15 ml) of (1.82 mmol) was added under ice cooling and stirred for 2 hours. Then, it stirred at room temperature for 22 hours. The obtained reaction solution was filtered, and the filtrate was distilled off under reduced pressure, followed by purification with LH-20 (eluent: methanol). The material thus obtained is referred to as M-PEG 550 -NPC. The reaction formula is shown below.
Ｍ−ＰＥＧ550−ＤＬ−Ｇ２−２Ｃ１８の合成
ＤＬ−Ｇ２−２Ｃ１８（０．１ｇ、０．０７６ｍｍｏｌ）をＤＭＳＯ（５ｍｌ）に溶解し、これにＭ−ＰＥＧ550−ＮＰＣ（０．４５ｇ、０．６３ｍｍｏｌ）のＤＭＳＯ溶液（７ｍｌ）を加え、室温で５日間攪拌した。その後、２４時間透析（Ｍｗ Ｃｕｔｏｆｆ １０００）を行い、凍結乾燥したのち、ＬＨ−２０（溶離液：メタノール）により精製した。
Synthesis of M-PEG 550 -DL-G2-2C18 DL-G2-2C18 (0.1 g, 0.076 mmol) was dissolved in DMSO (5 ml), and M-PEG 550 -NPC (0.45 g, 0. 63 mmol) in DMSO (7 ml) was added and stirred at room temperature for 5 days. Thereafter, dialysis (Mw Cutoff 1000) was performed for 24 hours, freeze-dried, and purified by LH-20 (eluent: methanol).
図６および図７に1ＨＮＭＲと13ＣＮＭＲの結果を示す。図６にはＮＭＲのチャートと構造式を示し、図７には、構造式とＰＥＧで修飾する前（Ａ）と修飾後（Ｂ）のチャートを示す。また図７では、３０から５０ｐｐｍまでの拡大図も記載した。拡大図では図面上が（Ａ）に対応し、図面下が（Ｂ）に対応する。図６および図７のデータを示す。括弧中の番号はそれぞれの図に示した構造の番号に対応する。 6 and 7 show the results of 1 HNMR and 13 CNMR. FIG. 6 shows an NMR chart and a structural formula, and FIG. 7 shows a structural formula and a chart before (A) and after modification (B) with PEG. FIG. 7 also shows an enlarged view from 30 to 50 ppm. In the enlarged view, the top of the drawing corresponds to (A), and the bottom of the drawing corresponds to (B). The data of FIG. 6 and FIG. 7 are shown. The numbers in parentheses correspond to the structure numbers shown in each figure.
1HNMR (CDCl3): δ 0.88 (1), δ 1.25 (2) and 1.53 (3), δ 2.35 (4, 6, 11 and 16), δ 2.53 (9 and 14), δ 2.72 (5, 10 and 15), δ 3.28 and 3.33 (8, 13, 18 and 19), δ 3.38 (23), δ 3.64 (22), δ 4.18 (21), δ 6.19 (20), δ 7.66 (7, 12 and 17);
13C NMR (CDCl3): δ 14.1 (1), δ 22.7, 27.3, 29.3, 29.4, 29.6, 29.7 and 31.9 (2 and 3), δ 34.1 (6, 11 and 16), δ 37.5 and 37.7 (8 and 13), δ 39.5 (19), δ 40.8 (18), δ 50.1 and 50.4 (9, 10, 14 and 15), δ 52.6 and 52.9 (4 and 5), δ 59.0 (23), δ 61.6, 63.8, 69.6, 70.4, 70.5, 70.8 and 71.9 (21 and 22), δ 156.9 (20), δ 172.7 and 173.3 (7, 12 and 17).
1 HNMR (CDCl 3 ): δ 0.88 (1), δ 1.25 (2) and 1.53 (3), δ 2.35 (4, 6, 11 and 16), δ 2.53 (9 and 14), δ 2.72 (5, 10 and 15), δ 3.28 and 3.33 (8, 13, 18 and 19), δ 3.38 (23), δ 3.64 (22), δ 4.18 (21), δ 6.19 (20), δ 7.66 (7, 12 and 17 );
13 C NMR (CDCl 3 ): δ 14.1 (1), δ 22.7, 27.3, 29.3, 29.4, 29.6, 29.7 and 31.9 (2 and 3), δ 34.1 (6, 11 and 16), δ 37.5 and 37.7 (8 and 13), δ 39.5 (19), δ 40.8 (18), δ 50.1 and 50.4 (9, 10, 14 and 15), δ 52.6 and 52.9 (4 and 5), δ 59.0 (23), δ 61.6, 63.8 , 69.6, 70.4, 70.5, 70.8 and 71.9 (21 and 22), δ 156.9 (20), δ 172.7 and 173.3 (7, 12 and 17).
図６の1ＨＮＭＲより、ジオクタデシル基末端のメチルに由来する０．８８ｐｐｍのピークの積分値を基準として、３．４ｐｐｍＰＥＧ末端（２３）のＯＣＨ3の積分値が理論値とほぼ一致したこと、さらに図７の13ＣＮＭＲより、デンドロン末端のメチレンプロトン１８，１９が高磁場シフトしたことから、ＤＬ−Ｇ２の全末端にＰＥＧが結合していると判断できる。また、ニンヒドリン反応が陰性であったことからも一級アミノ基は存在しないことがわかった。すなわち、ＰＥＧ550−を４本結合したＭ―ＰＥＧ−ＤＬ−Ｇ２が合成できた。
From the 1 HNMR of FIG. 6, the integrated value of OCH 3 at the 3.4 ppm PEG end (23) almost coincided with the theoretical value based on the integrated value of the 0.88 ppm peak derived from methyl at the end of the dioctadecyl group, Further, from 13 CNMR in FIG. 7, since the
図８にはＭ―ＰＥＧ−ＤＬ−Ｇ２単体であるリポソームと、DNAを加えたリポプレックスにした場合での粒径とPEG含有量の関係を示す。粒径は動的光散乱によって３回測定の平均値をプロットし、測定幅を縦線でしめした。縦軸は平均粒径、横軸はＰＥＧの割合を示す。黒四角はリポプレックスを示し、白丸はリポソームを示す。リポプレックスはＮ／Ｐ比が４であり、ＤＬ−Ｇ３−２Ｃ１２とＤＯＰＥとＰＥＧ550−ＤＬ−Ｇ２−２Ｃ１８を用いたものである。またDNAはホタルのルシファーゼ遺伝子を含むｐＣＭＶＬｕｃを用いた。 FIG. 8 shows the relationship between the particle size and the PEG content when the liposome is M-PEG-DL-G2 alone and the lipoplex is added with DNA. For the particle size, the average value of three measurements was plotted by dynamic light scattering, and the measurement width was shown by a vertical line. The vertical axis represents the average particle size, and the horizontal axis represents the ratio of PEG. Black squares indicate lipoplexes, and white circles indicate liposomes. The lipoplex has an N / P ratio of 4, and uses DL-G3-2C12, DOPE, and PEG 550 -DL-G2-2C18. As the DNA, pCMVLuc containing a firefly luciferase gene was used.
図８より、リポソームの状態であるとPEGの含有量は、平均粒径に影響を与えないが、リポプレックスになると、ＰＥＧ含有量が１．０ｍｏｌ％以上で粒径はほぼ２００ｎｍ〜２５０ｎｍに小さくなっていることがわかる。このようにPEGで修飾したデンドロン脂質を含有させることによって、リポプレックスにしたときの凝集を抑え、安定化させることができる。 From FIG. 8, the content of PEG does not affect the average particle size in the liposome state, but when it becomes lipoplex, the PEG content is 1.0 mol% or more and the particle size is almost 200 nm to 250 nm. You can see that By containing the dendron lipid modified with PEG in this way, aggregation when it is made into a lipoplex can be suppressed and stabilized.
本発明は、遺伝子治療において治療用のＤＮＡを細胞内に送り込むベクターとして利用でき、特に末端にリガンドを付加するなどによって標的細胞に対する特異性を持たせることができるため、ＤＤＳ（Ｄｒｕｇ Ｄｅｌｉｖｅｒｙ Ｓｙｓｔｅｍ）として利用することができる。 The present invention can be used as a vector for delivering therapeutic DNA into cells in gene therapy. In particular, since it can be made specific for a target cell by adding a ligand to the terminal or the like, as DDS (Drug Delivery System) Can be used.
Claims (4)
ＤＬ−Ｇ１：Ｒ1Ｒ2ＮＸ（ＸＨ2）2
ＤＬ−Ｇ２：Ｒ1Ｒ2ＮＸ（Ｘ（ＸＨ2）2）2
ＤＬ−Ｇ３：Ｒ1Ｒ2ＮＸ（Ｘ（Ｘ（ＸＨ2）2）2）2
ＤＬ−Ｇ４：Ｒ1Ｒ2ＮＸ（Ｘ（Ｘ（Ｘ（ＸＨ2）2）2）2）2
ＤＬ−Ｇ５：Ｒ1Ｒ2ＮＸ（Ｘ（Ｘ（Ｘ（Ｘ（ＸＨ2）2）2）2）2）2
ＤＬ−Ｇ６：Ｒ1Ｒ2ＮＸ（Ｘ（Ｘ（Ｘ（Ｘ（Ｘ（ＸＨ2）2）2）2）2）2）2
ＤＬ−Ｇ７：Ｒ1Ｒ2ＮＸ（Ｘ（Ｘ（Ｘ（Ｘ（Ｘ（Ｘ（ＸＨ2）2）2）2）2）2）2）2
ＤＬ−Ｇ８：Ｒ1Ｒ2ＮＸ（Ｘ（Ｘ（Ｘ（Ｘ（Ｘ（Ｘ（Ｘ（ＸＨ2）2）2）2）2）2）2）2）2
（式中 Ｒ1及びＲ2は、同一または異なったアルキル基、アルコキシ基、アリール基またはアラルキル基を示す。Ｘは、−ＣＨ2ＣＨ2ＣＯＮＨＣＨ2ＣＨ2Ｎ＜を示す。Ｒ3は糖類の残基を示す。Ｒ4はポリオキシエチレン基またはポリビニルアルコール基または、ポリビニルピロリドン基を示す。） A gene transport medium composition comprising a compound in which at least one of the XH 2 moieties of a compound represented by any one of the following formulas DL-G1 to DL-G8 is replaced with either XHR 3 or XHR 4 .
DL-G1: R 1 R 2 NX (XH 2 ) 2
DL-G2: R 1 R 2 NX (X (XH 2 ) 2 ) 2
DL-G3: R 1 R 2 NX (X (X (XH 2 ) 2 ) 2 ) 2
DL-G4: R 1 R 2 NX (X (X (X (XH 2 ) 2 ) 2 ) 2 ) 2
DL-G5: R 1 R 2 NX (X (X (X (X (XH 2 ) 2 ) 2 ) 2 ) 2 ) 2
DL-G6: R 1 R 2 NX (X (X (X (X (X (XH 2) 2) 2) 2) 2) 2) 2
DL-G7: R 1 R 2 NX (X (X (X (X (X (X (XH 2 ) 2 ) 2 ) 2 ) 2 ) 2 ) 2 ) 2 ) 2
DL-G8: R 1 R 2 NX (X (X (X (X (X (X (X (XH 2 ) 2 ) 2 ) 2 ) 2 ) 2 ) 2 ) 2 ) 2
(Wherein R 1 and R 2 represent the same or different alkyl group, alkoxy group, aryl group or aralkyl group. X represents —CH 2 CH 2 CONHCH 2 CH 2 N <. R 3 represents a saccharide. R 4 represents a residue, and R 4 represents a polyoxyethylene group, a polyvinyl alcohol group, or a polyvinyl pyrrolidone group.)
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