JP6137894B2 - Liposome-exosome hybrid vesicle and preparation method thereof - Google Patents
Liposome-exosome hybrid vesicle and preparation method thereof Download PDFInfo
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- JP6137894B2 JP6137894B2 JP2013060148A JP2013060148A JP6137894B2 JP 6137894 B2 JP6137894 B2 JP 6137894B2 JP 2013060148 A JP2013060148 A JP 2013060148A JP 2013060148 A JP2013060148 A JP 2013060148A JP 6137894 B2 JP6137894 B2 JP 6137894B2
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Landscapes
- Medicinal Preparation (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
本発明は、リポソーム−エキソソームハイブリッドベシクル及びその調製法、物質導入用担体、導入剤に関する。 The present invention relates to a liposome-exosome hybrid vesicle, a preparation method thereof, a substance introduction carrier, and an introduction agent.
細胞は、生体膜構造を有する様々なベシクル(extracellular vesicle)を放出している。その中で、エキソソームは、細胞が分泌するエンドソーム由来の50-100nm のサイズを有するベシクルであり、近年、エキソソーム中にメッセンジャーRNA(mRNA)やマイクロRNA(miRNA)が含まれており、他の細胞にRNA を運ぶシャトルとして機能することが見いだされた。生物における細胞間コミュニケーション手段として、これまでは細胞間の直接の接着や増殖因子、サイトカインなどの生理活性タンパク質、ペプチド、ホルモンを用いる経路が知られていたが、特定の細胞由来の細胞膜タンパク質や核酸を含むエキソソームが、長距離の細胞間コミュニケーション経路として、種々の生命現象に重要に関わっていることが明らかにされつつあり、医療やバイオへの応用研究が進められている。 Cells release various extracellular vesicles having a biological membrane structure. Among them, exosomes are vesicles with a size of 50-100 nm derived from endosomes secreted by cells. In recent years, exosomes contain messenger RNA (mRNA) and microRNA (miRNA) and other cells. Has been found to function as a shuttle to carry RNA. As a means of cell-to-cell communication in living organisms, until now, direct adhesion between cells and pathways using bioactive proteins such as growth factors and cytokines, peptides and hormones have been known, but cell membrane proteins and nucleic acids derived from specific cells It is becoming clear that exosomes, including selenium, are importantly involved in various life phenomena as long-distance intercellular communication pathways.
エキソソームをドラッグデリバリーキャリアとして応用した例はまだそれほど多くはないが、エキソソームを分泌する側の細胞を適切に選択することにより、また、疾病特異的に発現している膜タンパク質等のリガンドをエンドソームや細胞膜に過剰発現させてエキソソームに移行させるにより、標的指向性を上昇させ、ドラッグキャリアとして利用することが試みられている。例えば、Zhang らは、マウスリンパ腫EL-4 由来のエキソソームにがん細胞の増殖を抑制するクルクミンを含有させ、マウスの骨髄細胞に到達させることに成功している(非特許文献1)。また、Erviti らは、siRNAを搭載したエキソソームをマウスの脳に送り込むことに成功している(非特許文献2)。Erviti らは、脳にターゲティングするために、遺伝子工学的手法を用いてニューロン特異的ペプチド(RVG ペプチド)を融合させたエキソソーム膜タンパク質(Lamp2b)を発現する樹状細胞を作製した。その細胞から回収したエキソソームに電気穿孔法を用いてsiRNA を内包させ、マウスに投与したところ、脳のニューロンやミクログリアなどにsiRNA が送達され、標的遺伝子がノックダウンされることを報告している。また、Erviti らは、アルツハイマー病治療の標的遺伝子であるBACE1 のノックダウンにも成功している。また、東京医科大学の大野らのグループでは、上皮成長因子受容体[EGFR]に高い親和性を持つ人工ペプチド[GE11]を発現する遺伝子と腫瘍抑制性マイクロRNA を導入した細胞から抽出精製したエキソソームに、GE11 が膜表面に発現しており、目的のマイクロRNA が内包されていることを示した。さらにそこで得られたエキソソームをマウスに導入したところ、GE11 提示型エキソソームが乳がんモデルマウスで腫瘍の発達を効率よく抑制することを報告した。細胞種によりエキソソームはレセプター/リガンド相互作用、マクロピノサイトーシス等の様々なエンドサイトーシスやファゴサイトーシスによって受け手側の細胞に取り込まれると報告されている(非特許文献3)。このような背景から、エキソソームを用いたドラッグデリバリーは、新しい治療法として期待されている。 There are not many examples of applying exosomes as drug delivery carriers, but by appropriately selecting cells that secrete exosomes, ligands such as membrane proteins expressed specifically for diseases can also be used as endosomes and Attempts have been made to increase the target directivity by using it as a drug carrier by overexpressing it in the cell membrane and transferring it to the exosome. For example, Zhang et al. Have succeeded in allowing mouse lymphocytes EL-4-derived exosomes to contain curcumin that suppresses the growth of cancer cells to reach mouse bone marrow cells (Non-patent Document 1). Erviti et al. Have succeeded in sending an exosome loaded with siRNA into the mouse brain (Non-patent Document 2). Erviti et al. Created dendritic cells expressing exosomal membrane protein (Lamp2b) fused with neuron-specific peptide (RVG peptide) using genetic engineering techniques to target the brain. It has been reported that siRNA is encapsulated in exosomes collected from the cells using electroporation and administered to mice, and siRNA is delivered to brain neurons, microglia, etc., and the target gene is knocked down. Erviti and colleagues have also succeeded in knocking down BACE1, the target gene for the treatment of Alzheimer's disease. Ono's group at Tokyo Medical University also extracted and purified exosomes from cells transfected with a gene that expresses an artificial peptide [GE11] with high affinity for epidermal growth factor receptor [EGFR] and tumor suppressor microRNA. In addition, GE11 was expressed on the membrane surface, indicating that the target microRNA was encapsulated. Furthermore, when the obtained exosome was introduced into mice, it was reported that GE11-presenting exosomes efficiently suppressed tumor development in breast cancer model mice. It has been reported that exosomes are incorporated into recipient cells by various endocytosis and phagocytosis such as receptor / ligand interaction, macropinocytosis, etc. depending on the cell type (Non-patent Document 3). Against this background, drug delivery using exosomes is expected as a new therapeutic method.
一方、生体構成成分であるリン脂質から成るリポソームを用いたドラッグデリバリーは、近年盛んに研究されている。リポソームをドラッグキャリアとして用いる利点として、内包物質の制御のしやすさが挙げられる。リポソームを利用したドラッグキャリアは、PEG を修飾すること等により、血中滞留性の向上や補体等による捕捉から回避可能になり、また、標的細胞志向型の抗体等を表面に修飾することにより、標的特異性が飛躍的に向上している。近年本発明者らは、無細胞タンパク質合成系にリポソームを共存させることで、膜タンパク質の遺伝子発現に伴うリポソームへの直接構成、さらに、膜タンパク質を発現したリポソームだけを精製するシステムの構築に成功している(非特許文献4)。 On the other hand, drug delivery using liposomes composed of phospholipids, which are biological components, has been actively studied in recent years. An advantage of using liposomes as a drug carrier is ease of control of the inclusion substance. Drug carriers using liposomes can be improved from retention by increasing the retention in the blood or captured by complements, etc. by modifying PEG, and by modifying the surface of target cell-oriented antibodies, etc. , The target specificity has improved dramatically. In recent years, the present inventors have succeeded in constructing a system for purifying only liposomes expressing a membrane protein by directly constructing them into a liposome accompanying gene expression of a membrane protein by coexisting liposomes in a cell-free protein synthesis system. (Non-Patent Document 4).
特許文献1は、組換え体センダイウイルスを利用した膜融合性リポソームを開示しているが、エキソソームについての記載はない。特許文献2は、B型肝炎ウイルスタンパク質中空バイオナノ粒子とリポソームを用いたsiRNAの内包と細胞選択的なsiRNAの導入方法を開示しているが、エキソソームについての記載はない。 Patent Document 1 discloses a fusogenic liposome using a recombinant Sendai virus, but does not describe exosomes. Patent Literature 2 discloses a method for introducing siRNA and cell-selective siRNA using hepatitis B virus protein hollow bio-nanoparticles and liposomes, but there is no description of exosomes.
特許文献3は、外因性遺伝物質を取り込ませたエキソソームを含む組成物を開示し、エキソソームに遺伝物質を取り込ませる具体的方法としてエレクトロポレーションが開示されている。特許文献3の明細書には、トランスフェクション剤を使用できること、トランスフェクション剤としてカチオン性リポソームが例示されているが、カチオン性リポソームを使用した実施例の記載はない。 Patent Document 3 discloses a composition containing an exosome into which exogenous genetic material is incorporated, and electroporation is disclosed as a specific method for incorporating the genetic material into the exosome. The specification of Patent Document 3 exemplifies that transfection agents can be used and cationic liposomes as transfection agents, but there is no description of examples using cationic liposomes.
本発明は、エキソソームへの生理活性物質の導入の手法を提供することを目的とする。 An object of the present invention is to provide a technique for introducing a physiologically active substance into an exosome.
本発明は、以下のリポソーム−エキソソームハイブリッドベシクル及びその調製法、物質導入用担体、導入剤に関する。
項1. 生理活性物質を内包するリポソームとエキソソームを複合化してなる、リポソーム−エキソソームハイブリッドベシクル。
項2. 生理活性物質がタンパク質、核酸または薬物である項1に記載のベシクル。
項3. 前記リポソームがカチオン性リポソームである、項1又は2に記載のベシクル。
項4. 前記ベシクルがsiRNAを含む、項1〜3のいずれかに記載のベシクル。
項5. 項1〜4のいずれかに記載のベシクルからなる物質導入用担体。
項6. 項5に記載のベシクルからなる生理活性物質の導入剤。
項7. 生理活性物質を内包するリポソームとエキソソームを混合し、凍結・融解工程に供することを特徴とする、項1〜4のいずれかに記載のリポソーム−エキソソームハイブリッドベシクルの調製法。
項8. 生理活性物質を内包するカチオン性リポソームとエキソソームを混合し、複合化させることを特徴とする、項1〜4のいずれかに記載のリポソーム−エキソソームハイブリッドベシクルの調製法。
項9. 生理活性物質を内包するリポソームとエキソソームを混合し、ポリエチレングリコール(PEG)及び/又はPEG-脂質により複合化させることを特徴とする、項1〜4のいずれかに記載のリポソーム−エキソソームハイブリッドベシクルの調製法。
The present invention relates to the following liposome-exosome hybrid vesicle, a method for preparing the same, a carrier for substance introduction, and an introduction agent.
Item 1. A liposome-exosome hybrid vesicle formed by complexing a liposome encapsulating a physiologically active substance and an exosome.
Item 2. Item 2. The vesicle according to Item 1, wherein the physiologically active substance is a protein, nucleic acid, or drug.
Item 3. Item 3. The vesicle according to Item 1 or 2, wherein the liposome is a cationic liposome.
Item 4. Item 4. The vesicle according to any one of Items 1 to 3, wherein the vesicle comprises siRNA.
Item 5. Item 5. A substance introduction carrier comprising the vesicle according to any one of Items 1 to 4.
Item 6. Item 6. An agent for introducing a physiologically active substance comprising the vesicle according to Item 5.
Item 7. Item 5. The method for preparing a liposome-exosome hybrid vesicle according to any one of Items 1 to 4, wherein a liposome encapsulating a physiologically active substance and an exosome are mixed and subjected to a freezing / thawing step.
Item 8. Item 5. The method for preparing a liposome-exosome hybrid vesicle according to any one of Items 1 to 4, wherein a cationic liposome encapsulating a physiologically active substance and an exosome are mixed and complexed.
Item 9. Item 5. The liposome-exosome hybrid vesicle according to any one of Items 1 to 4, wherein a liposome encapsulating a physiologically active substance and an exosome are mixed and complexed with polyethylene glycol (PEG) and / or PEG-lipid. Preparation method.
本発明によれば、エキソソームに任意の生理活性物質を導入することができる。 According to the present invention, any physiologically active substance can be introduced into an exosome.
リポソームとエキソソームを複合化することにより、リポソームに内包した物質をエキソソームにも内包できる。リポソームは容易に膜組成を変えることができるため、本来エキソソームには内包不可能な分子の内包が可能になる。リポソームは、薬剤の電荷によって膜成分を選択することにより、高い封入効率で薬剤を封入可能である。また、リポソームのサイズをコントロールすることで、エキソソーム-リポソーム複合化物の最終的な大きさを制御することも可能である。エキソソームの構成成分を提示したカプセルの中に、プラスミドDNA を内封することも可能である。PEGで修飾されたリン脂質やコレステロール類は、脂質部分がエキソソームと相互作用し、PEG鎖がエキソソーム表面を覆うことで免疫系により攻撃されなくなり長期血中滞留性となる、いわゆるステルスーエキソソームを構築することができる。さらに、我々が開発したリポソームへの膜タンパク質直接構成法を用いて作成した、膜タンパク質発現リポソームと複合化させることにより、細胞のトランスフェクションを介すことなくエキソソームの膜の改変や修飾を行うことが可能である。化学修飾リポソームも加味すると、バリエーションは無限大であり、内包物と外膜をより生体適合性を高め、テイラーメイド医療に応用可能なレベルへと改変することが可能である。また、大きなサイズの一枚膜リポソーム(以下、一枚膜リポソームを「SUV」ということもある。)と複合化させることにより、簡単に基板上にエキソソーム上のタンパク質のマッピングが可能になる。 By complexing liposomes and exosomes, substances encapsulated in liposomes can also be encapsulated in exosomes. Since liposomes can easily change the membrane composition, it is possible to encapsulate molecules that cannot originally be encapsulated in exosomes. Liposomes can encapsulate drugs with high encapsulation efficiency by selecting membrane components according to the charge of the drug. It is also possible to control the final size of the exosome-liposome complex by controlling the size of the liposome. It is also possible to encapsulate the plasmid DNA in a capsule that displays exosome components. Phospholipids and cholesterols modified with PEG construct so-called stealth exosomes, in which the lipid part interacts with the exosome and the PEG chain covers the exosome surface so that it is not attacked by the immune system and becomes long-term blood retention. can do. In addition, by modifying the membrane protein-expressing liposomes created using the membrane protein direct construction method we have developed, we can modify or modify the exosome membrane without transfection of cells. Is possible. Including chemically modified liposomes, the variations are infinite, and the inclusion and outer membrane can be further improved to biocompatibility and modified to a level applicable to tailor-made medicine. In addition, by complexing with large-sized unilamellar liposomes (hereinafter, unilamellar liposomes may be referred to as “SUV”), it becomes possible to easily map the protein on the exosome on the substrate.
1つの実施形態において、本発明は、リポソームとエキソソームが複合化したリポソーム−エキソソームハイブリッドベシクルを提供するものである。 In one embodiment, the present invention provides a liposome-exosome hybrid vesicle in which a liposome and an exosome are complexed.
本発明におけるエキソソームとは、細胞から放出される小胞を広く含む。エキソソームの直径は30〜200nm程度、好ましくは30〜100nm程度であり、リン脂質、コレステロ−ルなどの脂質、タンパク質等を含む。エキソソームはそれを産生する限り、いかなる動物種または植物種由来のものであってもよい。動物種は、例えば、脊椎動物(例えば、ヒト、マウス、ラット、サル、イヌ、ネコ、ウシ、ウマ、ブタ、ラット、マウス、ハムスタ−、ウサギ、ヤギ、ニワトリ、サケ、マグロ等)が挙げられるが、好ましくは、薬物、核酸などの生理活性物質を導入する標的細胞と同種の動物由来であり、標的細胞が生体内の細胞である場合には、標的細胞と同じ動物に由来する小胞が好ましい。また、エキソソームが由来する細胞種は特に限定されないが、例えば、腫瘍細胞、樹状細胞、マクロファ−ジ、T細胞、B細胞、血小板、網状赤血球、上皮細胞、線維芽細胞、幹細胞、iPS細胞等の様々な種類の細胞を挙げることができる。また、エキソソームは種々の体液、例えば、血液、尿、腹水などから調製することもできる。 The exosome in the present invention broadly includes vesicles released from cells. The diameter of the exosome is about 30 to 200 nm, preferably about 30 to 100 nm, and includes phospholipids, lipids such as cholesterol, proteins, and the like. The exosome may be derived from any animal or plant species as long as it produces it. Examples of animal species include vertebrates (eg, humans, mice, rats, monkeys, dogs, cats, cows, horses, pigs, rats, mice, hamsters, rabbits, goats, chickens, salmon, tuna, etc.). However, preferably, when a target cell into which a physiologically active substance such as a drug or nucleic acid is introduced is derived from the same animal, and the target cell is an in vivo cell, a vesicle derived from the same animal as the target cell preferable. The cell type from which the exosome is derived is not particularly limited. For example, tumor cells, dendritic cells, macrophages, T cells, B cells, platelets, reticulocytes, epithelial cells, fibroblasts, stem cells, iPS cells, etc. Various types of cells can be mentioned. Exosomes can also be prepared from various body fluids such as blood, urine, ascites.
リポソームは、多重層リポソーム、一枚膜リポソームのいずれであってもよい。リポソームの大きさは40nm〜100μm程度、好ましくは50nm〜50μm程度、特に60nm〜10μm程度である。リポソームは、通常のナノメートルサイズのリポソームとジャイアントリポソームのいずれを使用してもよい。ジャイアントリポソームのサイズは通常1〜100マイクロメーター程度である。通常のナノメートルサイズのリポソームの大きさは、40nm〜300nm程度、好ましくは50nm〜200nm程度、特に60nm〜150nm程度である。リポソームのサイズ(粒子径)は、エクストルーダーを用いて、孔径の小さいフィルターを通過させることによって調節可能である。 The liposome may be either a multilamellar liposome or a monolayer liposome. The size of the liposome is about 40 nm to 100 μm, preferably about 50 nm to 50 μm, particularly about 60 nm to 10 μm. As the liposome, either a normal nanometer size liposome or a giant liposome may be used. The size of the giant liposome is usually about 1 to 100 micrometers. Ordinary nanometer-sized liposomes have a size of about 40 nm to 300 nm, preferably about 50 nm to 200 nm, particularly about 60 nm to 150 nm. The size (particle diameter) of the liposome can be adjusted by passing it through a filter having a small pore diameter using an extruder.
本明細書において、リポソームとエキソソームの「複合化」は、リポソームとエキソソームが融合する場合、リポソームとエキソソームが会合する場合の両方を含む。 As used herein, “complexation” of a liposome and an exosome includes both the case where the liposome and the exosome are fused, and the case where the liposome and the exosome are associated.
本明細書において、物質を「内包」するとは、リポソーム、エキソソームなどの各粒子の内部に物質が存在する場合と、リポソームとエキソソームが会合して1つの複合体を形成した場合にも、物質は、リポソーム、エキソソーム又はこれらの複合体に「内包」されている。 In the present specification, “encapsulation” of a substance means that the substance is present even when the substance exists inside each particle such as a liposome and exosome, and when the liposome and exosome associate to form one complex. , "Encapsulated" in liposomes, exosomes or complexes thereof.
本発明のハイブリッドベシクルは、リポソームとエキソソームが1:1で複合化(融合又は会合)したハイブリッドベシクルでもよく、1個のリポソームと複数のエキソソームが複合化(融合又は会合)したベシクルでもよく、複数のリポソームと1個のエキソソームが複合化(融合又は会合)したベシクルでもよく、複数のリポソームと複数のエキソソームが複合化(融合又は会合)したベシクルでもよい。 The hybrid vesicle of the present invention may be a hybrid vesicle in which liposomes and exosomes are complexed (fused or associated) 1: 1, or may be a vesicle in which one liposome and a plurality of exosomes are complexed (fused or associated). These vesicles may be complexed (fused or associated) with one exosome, or may be vesicles composed of a plurality of liposomes and a plurality of exosomes (fused or associated).
エキソソーム1個あたりに複合化するリポソームは、0.2〜5個程度、好ましくは0.3〜3個程度、より好ましくは0.5〜2個程度である。 The number of liposomes complexed per exosome is about 0.2 to 5, preferably about 0.3 to 3, more preferably about 0.5 to 2.
リポソームは超音波処理法、逆相蒸発法、凍結融解法、脂質溶解法、噴霧乾燥法などの従来公知の任意の方法により製造することができる。
リポソームの構成成分としては、リン脂質、コレステロール類、PEGで修飾されたリン脂質、PEGで修飾されたコレステロール類などが挙げられる。
Liposomes can be produced by any conventionally known method such as sonication, reverse phase evaporation, freeze-thaw, lipid lysis, or spray drying.
Examples of the constituent components of the liposome include phospholipids, cholesterols, phospholipids modified with PEG, and cholesterols modified with PEG.
リン脂質としては、ジパルミトイルホスファチジルエタノールアミン(DPPE)、ジステアロイルホスファチジルエタノールアミン(DSPE)などのホスファチジルエタノールアミン類;ジパルミトイルホスファチジルコリン(DPPC)、ジステアロイルホスファチジルコリン(DSPC)などのホスファチジルコリン類;ジパルミトイルホスファチジルセリン(DPPS)、ジステアロイルホスファチジルセリン(DSPS)などのホスファチジルセリン類;ジパルミトイルホスファチジン酸(DPPA)、ジステアロイルホスファチジン酸(DSPA)などのホスファチジン酸類、ジパルミトイルホスファチジルイノシトール(DPPI)、ジステアロイルホスファチジルイノシトール(DSPI)などのホスファチジルイノシトール類などが挙げられ、卵黄レシチン、大豆レシチン、リゾレシチン等の天然リン脂質、あるいはこれらを常法によって水素添加したものを使用することができる。 Phospholipids include phosphatidylethanolamines such as dipalmitoylphosphatidylethanolamine (DPPE) and distearoylphosphatidylethanolamine (DSPE); phosphatidylcholines such as dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC); dipalmitoylphosphatidyl Phosphatidyl serines such as serine (DPPS), distearoyl phosphatidyl serine (DSPS); phosphatidic acids such as dipalmitoyl phosphatidic acid (DPPA), distearoyl phosphatidic acid (DSPA), dipalmitoyl phosphatidylinositol (DPPI), distearoyl phosphatidylinositol Phosphatidylinosites such as (DSPI) Mentioned and Le acids, egg yolk lecithin, soybean lecithin, natural phospholipids lysolecithin, etc., or these can be used after hydrogenation by an ordinary method.
また、ポリエチレングリコール(PEG)で修飾された(PEG鎖を有する)リン脂質として、DSPE-PEG, mPEG(メトキシPEG)-DSPE(PEGの分子量、750, 1000, 2000, 5000,10000,20000, 30000, 40000)を使用することができる。 In addition, as a phospholipid modified with polyethylene glycol (PEG) (having a PEG chain), DSPE-PEG, mPEG (methoxyPEG) -DSPE (PEG molecular weight, 750, 1000, 2000, 5000,10000, 20000, 30000 , 40000) can be used.
コレステロール類としては、コレステロール(Chol)、3β−[N−(ジメチルアミノエタン)カルバモイル]コレステロール(DC−Chol)、N−(トリメチルアンモニオエチル)カルバモイルコレステロール(TC−Chol)などが挙げられる。
また、ポリエチレングリコール(PEG)で修飾された(PEG鎖を有する)コレステロール類として、Cholesterol-PEG, mPEG(メトキシPEG)- Cholesterol(PEGの分子量、1000, 2000, 5000,10000,20000, 30000, 40000)を使用することができる。
Examples of cholesterols include cholesterol (Chol), 3β- [N- (dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), N- (trimethylammonioethyl) carbamoylcholesterol (TC-Chol) and the like.
Further, as cholesterols modified with polyethylene glycol (PEG) (having PEG chain), Cholesterol-PEG, mPEG (methoxy PEG) -Cholesterol (molecular weight of PEG, 1000, 2000, 5000,10000, 20000, 30000, 40000) ) Can be used.
リポソームの構成成分は、これらの脂質を単独であるいは組み合わせて使用することができる。 As the constituent components of the liposome, these lipids can be used alone or in combination.
リポソームには、少なくとも1種のカチオン性脂質を包含させることができる。 The liposome can include at least one cationic lipid.
カチオン性脂質としては、DC-6-14(O,O’-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride)、DODAC(dioctadecyldimethylammonium chloride)、DOTMA(N-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium)、DDAB(didodecylammonium bromide)、DOTAP(1,2-dioleoyloxy-3-trimethylammonio propane)、DC-Chol(3β-N-(N',N',-dimethyl-aminoethane)-carbamol cholesterol)、DMRIE(1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium)、DOSPA(2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminum trifluoroacetate)等が挙げられる。 Cationic lipids include DC-6-14 (O, O'-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride), DODAC (dioctadecyldimethylammonium chloride), DOTMA (N- (2,3-dioleyloxy) propyl-N , N, N-trimethylammonium), DDAB (didodecylammonium bromide), DOTAP (1,2-dioleoyloxy-3-trimethylammoniopropane), DC-Chol (3β-N- (N ', N',-dimethyl-aminoethane) -carbamol cholesterol), DMRIE (1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium), DOSPA (2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminum trifluoroacetate) .
好ましい1つの実施形態において、本発明のリポソームは、カチオン性脂質、リン脂質、コレステロール類を含む。これらの比率としては、リポソーム全体を100重量部として
リン脂質:10〜100重量部、好ましくは30〜100重量部、より好ましくは70〜100重量部、
コレステロール類:0〜50重量部、好ましくは0〜30重量部、より好ましくは10〜20重量部。
カチオン性脂質:0〜80重量部、好ましくは10〜70重量部、より好ましくは30〜70重量部
である。
In one preferred embodiment, the liposome of the present invention comprises a cationic lipid, a phospholipid, and cholesterols. As these ratios, 100 parts by weight of the whole liposome, phospholipid: 10 to 100 parts by weight, preferably 30 to 100 parts by weight, more preferably 70 to 100 parts by weight,
Cholesterol: 0-50 parts by weight, preferably 0-30 parts by weight, more preferably 10-20 parts by weight.
Cationic lipid: 0 to 80 parts by weight, preferably 10 to 70 parts by weight, more preferably 30 to 70 parts by weight.
上記の比率において、リン脂質、コレステロール類の一部または全部がPEGで修飾されていてもよい。 In the above ratio, part or all of phospholipids and cholesterols may be modified with PEG.
リン脂質としては、DSPE-PG10G、DSPE-PEG350やPEG-コレステロールのようにPEGなどで修飾されたリン脂質やコレステロール類を使用することもできる。PEGで修飾されたリン脂質やコレステロール類は、脂質部分がエキソソームと相互作用し、PEG鎖がエキソソーム表面を覆うことで免疫系により攻撃されなくなり長期血中滞留性となる、いわゆるステルスーエキソソームを構築することができる。 As phospholipids, phospholipids and cholesterols modified with PEG such as DSPE-PG10G, DSPE-PEG350 and PEG-cholesterol can also be used. Phospholipids and cholesterols modified with PEG construct so-called stealth exosomes, in which the lipid part interacts with the exosome and the PEG chain covers the exosome surface so that it is not attacked by the immune system and becomes long-term blood retention. can do.
リポソームには、核酸、タンパク質、薬物などの生理活性物質を封入することで、エキソソームとの複合化時にこれらの生理活性物質をハイブリッドベシクルに導入することができる。 By encapsulating physiologically active substances such as nucleic acids, proteins and drugs in liposomes, these physiologically active substances can be introduced into hybrid vesicles when complexed with exosomes.
薬物は任意の薬物が使用され特に限定されないが、例えば、抗腫瘍剤、抗高血圧剤、抗低血圧剤、抗精神病剤、鎮痛剤、抗鬱剤、抗躁剤、抗不安剤、鎮静剤、催眠剤、抗癲癇剤、オピオイドアゴニスト、喘息治療剤、麻酔剤、抗不整脈剤、関節炎治療剤、鎮痙剤、ACEインヒビター、鬱血除去剤、抗生物質、抗狭心症剤、利尿剤、抗パーキンソン病剤、気管支拡張剤、抗利尿剤、利尿剤、抗高脂血症剤、免疫抑制剤、免疫調節剤、制吐剤、抗感染症剤、抗新生物剤、抗真菌剤、抗ウイルス剤、抗糖尿病剤、抗アレルギー剤、解熱剤、抗痛風剤、抗ヒスタミン剤、止痒剤、骨調節剤、心血管剤、コレステロール低下剤、抗マラリア剤、鎮咳剤、去痰剤、粘液溶解剤、鼻詰り用薬剤、ドパミン作動剤、消化管用薬剤、筋弛緩剤、神経筋遮断剤、副交感神経作動剤、プロスタグランジン、興奮薬、食欲抑制剤、甲状腺剤又は抗甲状腺剤、ホルモン、抗偏頭痛剤、抗肥満剤、抗炎症剤などとして作用し得るものが挙げられる。好ましい薬物は抗腫瘍剤である。抗腫瘍剤としては、ホルモン療法剤(例えば、ホスフェストロール、ジエチルスチルベストロール、クロロトリアニセリン、酢酸メドロキシプロゲステロン、酢酸メゲストロール、酢酸クロルマジノン、酢酸シプロテロン、ダナゾール、アリルエストレノール、ゲストリノン、メパルトリシン、ラロキシフェン、オルメロキフェン、レボルメロキシフェン、抗エストロゲン(例、クエン酸タモキシフェン、クエン酸トレミフェンなど)、ピル製剤、メピチオスタン、テストロラクトン、アミノグルテチイミド、LH−RHアゴニスト(例、酢酸ゴセレリン、ブセレリン、リュープロレリンなど)、ドロロキシフェン、エピチオスタノール、スルホン酸エチニルエストラジオール、アロマターゼ阻害薬(例、塩酸ファドロゾール、アナストロゾール、レトロゾール、エキセメスタン、ボロゾール、フォルメスタンなど)、抗アンドロゲン(例、フルタミド、ビカルタミド、ニルタミドなど)、5α−レダクターゼ阻害薬(例、フィナステリド、エプリステリドなど)、副腎皮質ホルモン系薬剤(例、デキサメタゾン、プレドニゾロン、ベタメタゾン、トリアムシノロンなど)、アンドロゲン合成阻害薬(例、アビラテロンなど)、レチノイドおよびレチノイドの代謝を遅らせる薬剤(例、リアロゾールなど)などが挙げられ、なかでもLH−RHアゴニスト(例、酢酸ゴセレリン、ブセレリン、リュープロレリンなど))、アルキル化剤(例えば、ナイトロジェンマスタード、塩酸ナイトロジェンマスタード−N−オキシド、クロラムブチル、シクロフォスファミド、イホスファミド、チオテパ、カルボコン、トシル酸インプロスルファン、ブスルファン、塩酸ニムスチン、ミトブロニトール、メルファラン、ダカルバジン、ラニムスチン、リン酸エストラムスチンナトリウム、トリエチレンメラミン、カルムスチン、ロムスチン、ストレプトゾシン、ピポブロマン、エトグルシド、カルボプラチン、シスプラチン、ミボプラチン、ネダプラチン、オキサリプラチン、アルトレタミン、アンバムスチン、塩酸ジブロスピジウム、フォテムスチン、プレドニムスチン、プミテパ、リボムスチン、テモゾロミド、トレオスルファン、トロフォスファミド、ジノスタチンスチマラマー、カルボコン、アドゼレシン、システムスチン、ビゼレシン)、代謝拮抗剤(例えば、メルカプトプリン、6−メルカプトプリンリボシド、チオイノシン、メトトレキサート、エノシタビン、シタラビン、シタラビンオクフォスファート、塩酸アンシタビン、5−FU系薬剤(例、フルオロウラシル、テガフール、UFT、ドキシフルリジン、カルモフール、ガロシタビン、エミテフールなど)、アミノプテリン、ロイコボリンカルシウム、タブロイド、ブトシン、フォリネイトカルシウム、レボフォリネイトカルシウム、クラドリビン、エミテフール、フルダラビン、ゲムシタビン、ヒドロキシカルバミド、ペントスタチン、ピリトレキシム、イドキシウリジン、ミトグアゾン、チアゾフリン、アンバムスチン)、抗癌性抗生物質(例えば、アクチノマイシンD、アクチノマイシンC、マイトマイシンC、クロモマイシンA3、塩酸ブレオマイシン、硫酸ブレオマイシン、硫酸ペプロマイシン、塩酸ダウノルビシン、塩酸ドキソルビシン、塩酸アクラルビシン、塩酸ピラルビシン、塩酸エピルビシン、ネオカルチノスタチン、ミスラマイシン、ザルコマイシン、カルチノフィリン、ミトタン、塩酸ゾルビシン、塩酸ミトキサントロン、塩酸イダルビシン)、植物由来抗癌剤(例えば、エトポシド、リン酸エトポシド、硫酸ビンブラスチン、硫酸ビンクリスチン、硫酸ビンデシン、テニポシド、パクリタキセル、ドセタクセル、ビノレルビン、カンプトテシン、塩酸イリノテカン)、免疫療法剤(BRM)(例えば、ピシバニール、クレスチン、シゾフィラン、レンチナン、ウベニメクス、インターフェロン、インターロイキン、マクロファージコロニー刺激因子、顆粒球コロニー刺激因子、エリスロポイエチン、リンホトキシン、BCGワクチン、コリネバクテリウムパルブム、レバミゾール、ポリサッカライドK、プロコダゾール)、細胞増殖因子ならびにその受容体の作用を阻害する薬剤(例えば、トラスツズマブ(ハーセプチン(商標);抗HER2抗体)、ZD1839(イレッサ)、グリーベック(GLEEVEC)などの抗体医薬)が挙げられる。抗腫瘍剤の対象となる癌の種類としては、結腸・直腸癌、肝臓癌、腎臓癌、頭頸部癌、食道癌、胃癌、胆道癌、胆のう・胆管癌、膵臓癌、肺癌、乳癌、卵巣癌、子宮頚癌、子宮体癌、膀胱癌、前立腺癌、精巣腫瘍、骨・軟部肉腫、白血病、悪性リンパ腫、多発性骨髄腫、皮膚癌、脳腫瘍等が挙げられ、好ましくは結腸・直腸癌、胃癌、頭頸部癌、肺癌、乳癌、膵臓癌、胆道癌、肝臓癌が挙げられる。 Any drug can be used and is not particularly limited. For example, antitumor agents, antihypertensive agents, antihypertensive agents, antipsychotic agents, analgesics, antidepressants, antidepressants, anxiolytics, sedatives, hypnosis Agent, antiepileptic agent, opioid agonist, asthma treatment agent, anesthetic agent, antiarrhythmic agent, arthritis treatment agent, antispasmodic agent, ACE inhibitor, decongestant, antibiotic, antianginal agent, diuretic agent, antiparkinsonian agent, Bronchodilators, antidiuretics, diuretics, antihyperlipidemic agents, immunosuppressants, immunomodulators, antiemetics, antiinfectives, antineoplastics, antifungals, antivirals, antidiabetics , Antiallergic agent, antipyretic agent, antigout agent, antihistamine, antidiarrheal agent, bone regulator, cardiovascular agent, cholesterol-lowering agent, antimalarial agent, antitussive agent, expectorant, mucolytic agent, nasal congestion agent, dopaminergic agent , Gastrointestinal drugs, muscle relaxants, neuromuscular blockers, Sympathomimetic agents, prostaglandins, stimulants, appetite suppressants, thyroid or antithyroid agent, hormone, antimigraine agents, anti-obesity agents include those that can act as such an anti-inflammatory agent. A preferred drug is an antitumor agent. Antitumor agents include hormonal therapeutic agents (eg, phosfestol, diethylstilbestrol, chlorotrianiserin, medroxyprogesterone acetate, megestrol acetate, chlormadinone acetate, cyproterone acetate, danazol, allylestrenol, gestrinone. , Mepartricin, raloxifene, olmeloxifen, levormeloxifene, antiestrogens (eg, tamoxifen citrate, toremifene citrate, etc.), pill formulations, mepithiostane, testrolactone, aminoglutethimide, LH-RH agonists (eg, goserelin acetate) , Buserelin, leuprorelin, etc.), droloxifene, epithiostanol, ethinyl estradiol sulfonate, aromatase inhibitors (eg, fadrozole hydrochloride, anastrozo) , Letrozole, exemestane, borozole, formestane, etc.), antiandrogens (eg, flutamide, bicalutamide, nilutamide, etc.), 5α-reductase inhibitors (eg, finasteride, epristeride, etc.), corticosteroids (eg, dexamethasone) , Prednisolone, betamethasone, triamcinolone, etc.), androgen synthesis inhibitors (eg, abiraterone), retinoids and drugs that delay the metabolism of retinoids (eg, riarosol), among others, LH-RH agonists (eg, goserelin acetate) , Buserelin, leuprorelin, etc.)), alkylating agents (eg, nitrogen mustard, nitrogen mustard hydrochloride-N-oxide, chlorambutyl, cyclophosphamide, ifosfamide, Thiotepa, carbocon, improsulfan tosylate, busulfan, nimustine hydrochloride, mitobronitol, melphalan, dacarbazine, ranimustine, sodium estramsine phosphate, triethylenemelamine, carmustine, lomustine, streptozocin, pipobroman, etoglucid, carboplatin, cisplatin, Miboplatin, nedaplatin, oxaliplatin, altretamine, ambermustine, dibrospium hydrochloride, fotemustine, prednimustine, pumitepa, ribomustine, temozolomide, treosulphane, trophosphamide, dinostatin timamarer, carbocon, adzelesin, systemustin, biselecin) Antimetabolites (eg mercaptopurine, 6-mercaptopurine riboside, thioino , Methotrexate, enositabine, cytarabine, cytarabine ocphosphatate, ancitabine hydrochloride, 5-FU drugs (eg, fluorouracil, tegafur, UFT, doxyfluridine, carmofur, garocitabine, emiteful, etc.), aminopterin, leucovorin calcium, tabloid Folinate calcium, levofolinate calcium, cladribine, emitefur, fludarabine, gemcitabine, hydroxycarbamide, pentostatin, piritrexim, idoxyuridine, mitoguazone, thiazofurin, ambmustine), anticancer antibiotics (eg, actinomycin D, actinomycin C, Mitomycin C, chromomycin A3, bleomycin hydrochloride, bleomycin sulfate, pepromy sulfate , Daunorubicin hydrochloride, doxorubicin hydrochloride, aclarubicin hydrochloride, pirarubicin hydrochloride, epirubicin hydrochloride, neocalcinostatin, misramicin, sarcomomycin, carcinophylline, mitotane, zorubicin hydrochloride, mitoxantrone hydrochloride, idarubicin hydrochloride), plant-derived anticancer agents (for example, , Etoposide, etoposide phosphate, vinblastine sulfate, vincristine sulfate, vindesine sulfate, teniposide, paclitaxel, docetaxel, vinorelbine, camptothecin, irinotecan hydrochloride, immunotherapeutic agent (BRM) (eg, picibanil, crestin, schizophyllan, fenibran, lentinone, ubenix Interleukin, macrophage colony stimulating factor, granulocyte colony stimulating factor, erythropoietin, lymphotoxin, BCG vaccine, Corynebacterium parvum, levamisole, polysaccharide K, procodazole), agents that inhibit the action of cell growth factors and their receptors (eg, trastuzumab (Herceptin ™; anti-HER2 antibody), ZD1839 (Iressa) And antibody drugs such as Gleevec). The types of cancer that are targeted by anti-tumor agents are colorectal cancer, liver cancer, kidney cancer, head and neck cancer, esophageal cancer, stomach cancer, biliary tract cancer, gallbladder / bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer. Cervical cancer, endometrial cancer, bladder cancer, prostate cancer, testicular tumor, bone / soft tissue sarcoma, leukemia, malignant lymphoma, multiple myeloma, skin cancer, brain tumor, etc., preferably colorectal cancer, stomach cancer , Head and neck cancer, lung cancer, breast cancer, pancreatic cancer, biliary tract cancer, liver cancer.
これらの薬剤、核酸、タンパク質などの生理活性物質は、1種単独で用いてもよく、2種以上を併用してもよい。 These physiologically active substances such as drugs, nucleic acids and proteins may be used alone or in combination of two or more.
核酸としては、特に制限はなく、DNA、RNA、DNAとRNAのキメラ核酸、DNA/RNAのハイブリッド等いかなるものであってもよい。また、核酸は1〜3本鎖のいずれも用いることができるが、好ましくは1本鎖又は2本鎖である。核酸は、プリンまたはピリミジン塩基のN−グリコシドであるその他のタイプのヌクレオチド、あるいは非ヌクレオチド骨格を有するその他のオリゴマー(例えば、市販のペプチド核酸(PNA)等)または特殊な結合を含有するその他のオリゴマー(但し、該オリゴマーはDNAやRNA中に見出されるような塩基のペアリングや塩基の付着を許容する配置をもつヌクレオチドを含有する)などであってもよい。さらには公知の修飾の付加されたもの、例えば当該分野で知られた標識のあるもの、キャップの付いたもの、メチル化されたもの、1個以上の天然のヌクレオチドを類縁物で置換したもの、分子内ヌクレオチド修飾のされたもの、例えば非荷電結合(例えば、メチルホスホネート、ホスホトリエステル、ホスホルアミデート、カルバメートなど)を持つもの、電荷を有する結合または硫黄含有結合(例えば、ホスホロチオエート、ホスホロジチオエートなど)を持つもの、例えば蛋白質(ヌクレアーゼ、ヌクレアーゼ・インヒビター、トキシン、抗体、シグナルペプチドなど)や糖(例えば、モノサッカライドなど)などの側鎖基を有しているもの、インターカレート化合物(例えば、アクリジン、プソラレンなど)を持つもの、キレート化合物(例えば、金属、放射活性をもつ金属、ホウ素、酸化性の金属など)を含有するもの、アルキル化剤を含有するもの、修飾された結合を持つもの(例えば、αアノマー型の核酸など)であってもよい。好ましい核酸としては、siRNAなどのRNAが挙げられる。 The nucleic acid is not particularly limited, and may be any DNA, RNA, chimeric nucleic acid of DNA and RNA, DNA / RNA hybrid, or the like. The nucleic acid can be any one of 1 to 3 strands, but is preferably a single strand or a double strand. Nucleic acids may be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or other oligomers having a non-nucleotide backbone (eg, commercially available peptide nucleic acids (PNA), etc.) or other oligomers containing special linkages (However, the oligomer contains a nucleotide having a configuration allowing base pairing or base attachment as found in DNA or RNA). Furthermore, those with known modifications, such as those with labels known in the art, capped, methylated, one or more natural nucleotides substituted with analogs, Intramolecular nucleotide modifications, such as those having uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg, phosphorothioates, phosphoro Dithioate, etc.), for example, proteins (nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, etc.) and sugars (eg, monosaccharides, etc.), side chain groups, intercalating compounds (Eg, acridine, psoralen, etc.), chelating Containing materials (eg, metals, radioactive metals, boron, oxidizing metals, etc.), containing alkylating agents, or having modified bonds (eg, α-anomeric nucleic acids) It may be. Preferred nucleic acids include RNA such as siRNA.
siRNAとは、標的遺伝子のmRNAもしくは初期転写産物のヌクレオチド配列又はその部分配列(好ましくはコード領域内)(初期転写産物の場合はイントロン部分を含む)に相同なヌクレオチド配列とその相補鎖からなる二本鎖オリゴRNAである。siRNAに含まれる、標的ヌクレオチド配列と相同な部分の長さは、通常、約18塩基以上、例えば約20塩基前後(代表的には約21〜23塩基長)の長さであるが、RNA干渉を引き起こすことが出来る限り、特に限定されない。また、siRNAの全長も、通常、約18塩基以上、例えば約20塩基前後(代表的には約21〜23塩基長)の長さであるが、RNA干渉を引き起こすことが出来る限り、特に限定されない。 The siRNA is a nucleotide sequence homologous to the nucleotide sequence of the target gene mRNA or the initial transcript or a partial sequence thereof (preferably within the coding region) (including an intron in the case of the initial transcript) and a complementary sequence thereof. This is a single-stranded oligo RNA. The length of a portion homologous to the target nucleotide sequence contained in siRNA is usually about 18 bases or more, for example, about 20 bases (typically about 21 to 23 bases) in length, but RNA interference Is not particularly limited as long as it can cause Further, the total length of siRNA is usually about 18 bases or more, for example, about 20 bases (typically about 21 to 23 bases in length), but is not particularly limited as long as it can cause RNA interference. .
標的ヌクレオチド配列と、siRNAに含まれるそれに相同な配列との関係については、100%一致していてもよいし、塩基の変異があってもよい(少なくとも70%、好ましくは80%、より好ましくは90%、最も好ましくは95%以上の同一性の範囲内であり得る)。 The relationship between the target nucleotide sequence and the sequence homologous to that contained in the siRNA may be 100% identical or may have base mutations (at least 70%, preferably 80%, more preferably 90%, most preferably within 95% identity range).
siRNAは、5’又は3’末端に5塩基以下、好ましくは2塩基からなる、塩基対を形成しない、付加的な塩基を有していてもよい。該付加的塩基は、DNAでもRNAでもよいが、DNAを用いるとsiRNAの安定性を向上させることができる。このような付加的塩基の配列としては、例えばug-3’、uu-3’、tg-3’、tt-3’、ggg-3’、guuu-3’、gttt-3’、ttttt-3’、uuuuu-3’等の配列が挙げられるが、これに限定されるものではない。 The siRNA may have an additional base at the 5 'or 3' end of 5 bases or less, preferably 2 bases, which does not form a base pair. The additional base may be DNA or RNA, but the use of DNA can improve the stability of siRNA. Examples of such additional base sequences include ug-3 ', uu-3', tg-3 ', tt-3', ggg-3 ', guuu-3', gttt-3 ', ttttt-3 Examples of the sequence include ', uuuuu-3', but are not limited thereto.
siRNAは任意の標的遺伝子に対するものであってよいが、本発明の核酸導入剤を疾患の予防・治療剤として用いる場合には、エキソソーム中に封入されるsiRNAは、その発現亢進が対象疾患の発症および/または増悪に関与する遺伝子を標的とするものであることが好ましく、より具体的には、その遺伝子に対するアンチセンス核酸が、臨床もしくは前臨床段階に進んでいる遺伝子や新たに知られた遺伝子を標的とするもの等が挙げられる。 The siRNA may be directed to any target gene. However, when the nucleic acid introduction agent of the present invention is used as a prophylactic / therapeutic agent for diseases, siRNA encapsulated in exosomes has an enhanced expression of the target disease. It is preferable that the target is a gene involved in exacerbation, and more specifically, the antisense nucleic acid for the gene is a clinically advanced or preclinical stage gene or a newly known gene And the like.
siRNAは、1種のみで使用してもよく、2種以上を組み合わせて使用してもよい。 siRNA may be used alone or in combination of two or more.
タンパク質としては、酵素、受容体、抗体、抗原、インターフェロン、インターロイキンなどのサイトカインなどが挙げられる。 Examples of the protein include enzymes, receptors, antibodies, antigens, cytokines such as interferon and interleukin.
本発明のハイブリッドベシクルの大きさは、100ナノメーター〜20マイクロメーター程度、好ましくは100〜200ナノメーター程度である。 The size of the hybrid vesicle of the present invention is about 100 nanometers to 20 micrometers, preferably about 100 to 200 nanometers.
リポソームとエキソソームのハイブリッドベシクルの製造法の例を具体的に説明すると、該リポソームとエキソソームを水などの適当な溶媒中で混合し、凍結・融解を繰り返すことにより、リポソームとエキソソームを複合化させることができる。凍結融解は、1〜30回程度、好ましくは10〜30回程度、より好ましくは20〜30回程度が挙げられる。 An example of a method for producing a liposome and exosome hybrid vesicle will be explained in detail. The liposome and exosome are mixed in an appropriate solvent such as water, and freeze-thaw is repeated to complex the liposome and exosome. Can do. Freezing and thawing may be performed 1 to 30 times, preferably about 10 to 30 times, and more preferably about 20 to 30 times.
リポソームとしてカチオン性リポソームを使用した場合、カチオン性リポソームとエキソソームを混合することにより複合化することができる。複合化は、反応温度4〜50℃程度、反応時間1〜24時間程度で実施することができる。 When a cationic liposome is used as the liposome, it can be complexed by mixing the cationic liposome and the exosome. The compounding can be carried out at a reaction temperature of about 4 to 50 ° C. and a reaction time of about 1 to 24 hours.
複合化反応は、エキソソーム 100重量部に対し、リポソームを20〜1000重量部程度、好ましくは100〜1000重量部程度使用する。 In the complexing reaction, the liposome is used in an amount of about 20 to 1000 parts by weight, preferably about 100 to 1000 parts by weight with respect to 100 parts by weight of the exosome.
複合化にPEGを使用する場合、生理活性物質を内包するリポソームとエキソソームをポリエチレングリコール(PEG)の存在下に混合し、複合化させてリポソーム−エキソソームハイブリッドベシクルを調製することができる。PEGは、0〜40重量%程度の濃度でリポソームとエキソソームを含む混合液中に加えることができる。 When PEG is used for conjugation, a liposome encapsulating a physiologically active substance and an exosome can be mixed in the presence of polyethylene glycol (PEG) and complexed to prepare a liposome-exosome hybrid vesicle. PEG can be added to a mixed solution containing liposomes and exosomes at a concentration of about 0 to 40% by weight.
使用されるPEGとしては、PEG500〜PEG10000、好ましくはPEG1000〜PEG8000が挙げられる。 Examples of PEG used include PEG500 to PEG10000, preferably PEG1000 to PEG8000.
また、DSPE-PG10G、DSPE-PEG350のようにPEGなどで修飾されたリン脂質、PEGで修飾されたコレステロール類などのPEG-脂質をエキソソームと複合化する場合には、PEG-脂質とエキソソーム中の脂質のモル比が0〜30モル%程度になるように加える。 When complexing PEG-lipids such as DSPE-PG10G and DSPE-PEG350 with PEG, such as phospholipids modified with PEG, and cholesterols modified with PEG, the PEG-lipid and exosome The lipid is added so that the molar ratio of lipid is about 0 to 30 mol%.
以下、本発明を実施例を用いてより詳細に説明するが、本発明が実施例に限定されないことはいうまでもない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, it cannot be overemphasized that this invention is not limited to an Example.
実施例1
<実験方法>
1. 凍結融解法による膜融合
1.1実験手法
1.1.1. 試薬
DOPS [1,2-dioleoyl-sn-glycero-3-phospho-L-serine]
Rhodamine-DHPE [Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt]
NBD-DHPE [N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- 1,2-dihexadecanoyl-sn- glycero-3- phosphoethanolamine, triethylammonium salt]
GelStar(登録商標)
Example 1
<Experiment method>
1. Membrane fusion by freeze-thaw method
1.1 Experimental method
1.1.1. Reagents
DOPS [1,2-dioleoyl-sn-glycero-3-phospho-L-serine]
Rhodamine-DHPE [Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt]
NBD-DHPE [N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3- phosphoethanolamine, triethylammonium salt]
GelStar (registered trademark)
1.1.2. SUV作製
クロロホルム:メタノール=2:1溶液に溶解したDOPSに、NBD-DHPEとRhodamine-DHPEが1%になるように添加し、アルゴンガス環流下で溶媒を蒸発させて脂質フィルムを作成した。作成した脂質フィルムにPBSバッファーを150μL加えて37℃で5分静置し、ボルテックスミキサーで撹拌した後エクストリューダーで100 nmのフィルターにかけてSUVを作成した。この時のDOPSの最終濃度が50 mMになるようにした。無色素のSUVも同様に作成し、コントロール実験に供した。
1.1.2. Preparation of SUV Chloroform: Methanol = 2: 1 To DOPS dissolved in a solution, add NBD-DHPE and Rhodamine-DHPE to 1% and evaporate the solvent under reflux of argon gas to form a lipid film. Created. 150 μL of PBS buffer was added to the prepared lipid film, allowed to stand at 37 ° C. for 5 minutes, stirred with a vortex mixer, and then applied to a 100 nm filter with an extruder to prepare an SUV. The final concentration of DOPS at this time was adjusted to 50 mM. A dye-free SUV was prepared in the same manner and subjected to a control experiment.
1.1.3. 細胞培養上清からのエキソソーム精製
K562細胞(ヒト白血病細胞株)、1.0~2.0×106個/mLの濃度でエキソソーム不含有培地(FBS由来のエキソソームを取り除いた培地)に懸濁し、15時間培養した後、細胞懸濁液を回収し、1000 rpmで10分遠心後、上清を回収しさらに12000g, 4℃で30分間遠心した。この上清を回収し100000g, 4℃で2時間遠心した。上清を捨て、5 mLのPBSを入れ、100000g, 4℃で2時間遠心した。遠心後、上清を捨てPBSで沈殿を再懸濁しK562由来エキソソーム懸濁液を得た。
1.1.3. Purification of exosomes from cell culture supernatant
K562 cells (human leukemia cell line), suspended in an exosome-free medium (medium from which FBS-derived exosomes have been removed) at a concentration of 1.0 to 2.0 × 10 6 cells / mL, cultured for 15 hours, After collecting and centrifuging at 1000 rpm for 10 minutes, the supernatant was recovered and further centrifuged at 12000 g at 4 ° C. for 30 minutes. The supernatant was collected and centrifuged at 100,000 g at 4 ° C. for 2 hours. The supernatant was discarded, 5 mL of PBS was added, and the mixture was centrifuged at 100,000 g at 4 ° C. for 2 hours. After centrifugation, the supernatant was discarded and the precipitate was resuspended with PBS to obtain a K562-derived exosome suspension.
RAW264.7細胞(マウスマクロファージ細胞株)を1×106個/mlの濃度でエキソソーム不含有培地2 Lに懸濁し、18〜24時間培養した後、細胞懸濁液を回収し、400 g, 4℃で10分遠心後、上清を回収しさらに10000 g、4℃で15分間遠心した。この上清を孔径0.45μm及び孔径0.22μmのフィルターで濾過後、限外濾過膜を用いて180 mlまで濃縮した。この濃縮上清を孔径0.8 μmのフィルターに通した後、100000 g、4℃で2時間遠心した。上清をアスピレーターで吸引除去後、エキソソームを含む沈査をPBSで洗浄し、100000 g、4℃で2時間遠心した。遠心後、上清をアスピレーターで吸引除去後、沈査を200 μlのPBSに再懸濁しRAW264.7由来エキソソーム懸濁液を得た。 RAW264.7 cells (mouse macrophage cell line) were suspended in 2 L of exosome-free medium at a concentration of 1 × 10 6 cells / ml and cultured for 18-24 hours. After centrifugation at 4 ° C for 10 minutes, the supernatant was recovered and further centrifuged at 10000 g and 4 ° C for 15 minutes. The supernatant was filtered through a filter having a pore size of 0.45 μm and a pore size of 0.22 μm, and then concentrated to 180 ml using an ultrafiltration membrane. The concentrated supernatant was passed through a filter having a pore size of 0.8 μm, and then centrifuged at 100000 g at 4 ° C. for 2 hours. The supernatant was removed by suction with an aspirator, and the precipitate containing exosomes was washed with PBS and centrifuged at 100,000 g for 2 hours at 4 ° C. After centrifugation, the supernatant was removed by suction with an aspirator, and the precipitate was resuspended in 200 μl of PBS to obtain a RAW264.7-derived exosome suspension.
1.1.4. 核酸染色剤を用いた複合化モニター実験
膜を透過しない核酸染色剤を封入したSUVをエキソソームに複合化させることで、内部溶液混合によるRNA検出を行った。150 μLで30 mM DOPSになる脂質フィルムに、37℃に暖めておいたGelStar(登録商標)10 μLとPBS140 μLを加え、5分程度37℃で膨潤させ、ボルテックスミキサーで撹拌した後、エクストリューダーで100 nmフィルターに通してSUVを作成した。その後、SUVを精製し、余剰色素を除去した。K562 エキソソーム(Lot32) 1.3 μgと、最終濃度が42.3 μMのDOPSsuv (including 9000x GelStar(登録商標))を混合し、蛍光強度を測定した。混合後に液体窒素で完全に固化した後室温にて20分以上静置した凍結融解法で処理したものの蛍光強度も測定した。また、コントロールとして、GelStar(登録商標)10 μLとPBS140 μLをゲル濾過したものを同じ要領用いたもので同様の実験を行なった。
1.1.4. Complexation monitor experiment using nucleic acid stain RNA detection by mixing internal solution was performed by complexing SUV encapsulating nucleic acid stain that does not permeate the membrane with exosome. To a lipid film that becomes 30 mM DOPS at 150 μL, add 10 μL of GelStar (registered trademark) warmed to 37 ° C. and 140 μL of PBS, swell at 37 ° C. for about 5 minutes, and stir with a vortex mixer. SUVs were made by passing them through a 100 nm filter. Then, SUV was refine | purified and the excess pigment | dye was removed. The fluorescence intensity was measured by mixing 1.3 μg of K562 exosome (Lot32) and DOPSsuv (including 9000x GelStar (registered trademark)) having a final concentration of 42.3 μM. After mixing, the solid was completely solidified with liquid nitrogen, and the fluorescence intensity of the sample treated by the freeze-thaw method was allowed to stand at room temperature for 20 minutes or more. As a control, the same experiment was performed using GelStar (registered trademark) 10 μL and PBS 140 μL gel-filtered in the same manner.
1.2. 結果と考察
最終濃度4 μMのDOPSsuv (1% NBD-PE, Rhodamine-PE) と50 μL中でタンパク質量5. 3 μgになるようにK562エキソソームを混合し、液体窒素で完全に固化と室温で完全に融解させることを15回繰り返して蛍光強度を測定した。
1.2. Results and Discussion Mix the final concentration of 4 μM DOPSsuv (1% NBD-PE, Rhodamine-PE) with 50 μL of K562 exosome to a protein content of 5.3 μg, and completely solidify with liquid nitrogen. Fluorescence intensity was measured by repeating complete melting at room temperature 15 times.
図1の緑色のラインが、エキソソームと混合直後のSUVの蛍光強度であり、薄紫色が、氷上で凍結融解にかかった時間と同等の時間を経過したサンプルの蛍光強度である。青色のラインが、凍結融解したものである。氷上静置したサンプルは、短波長側の蛍光強度が増加したが、長波長側の蛍光強度は下がらなかった。一方で、凍結融解したサンプルでは、長波長側の蛍光強度の減少がみられた。この結果をさらに詳しく知る為に、ナノサイトで凍結融解したものとしないものでのサイズを測定した。 The green line in FIG. 1 is the fluorescence intensity of the SUV immediately after mixing with the exosome, and the light purple color is the fluorescence intensity of the sample after a time equivalent to the time taken for freezing and thawing on ice. The blue line is frozen and thawed. The sample placed on ice increased the fluorescence intensity on the short wavelength side, but did not decrease the fluorescence intensity on the long wavelength side. On the other hand, in the frozen and thawed sample, a decrease in fluorescence intensity on the long wavelength side was observed. In order to know this result in more detail, the sizes of those that were frozen and thawed at nanosites were measured.
その結果、凍結融解したもの(凍結融解15回)ではサイズ分布が全体的に大きくなっており、FRET解消法と併せてエキソソームとリポソームが複合化していることが示唆された。 As a result, the size distribution of the freeze-thawed product (15 times of freeze-thaw) was increased overall, suggesting that exosomes and liposomes were complexed together with the FRET elimination method.
Raw エキソソームの場合
DIOとR18で共染色したエキソソーム(150 μg/mL)10 μLに、100 μMのリポソーム溶液を90 μL加え、液体窒素中で完全に固化し、37 ℃のヒートブロックにて溶解を5回繰り返す毎にサンプルの蛍光強度と粒子径の測定を行なった。また、同様にして作成したサンプルにTRITONX-100を1%添加し、完全に脂質混合が起こった条件での緑色の蛍光強度を100%とした時の各回数での蛍光強度を%としてFRET解消効率2(図4)を求めた。蛍光強度の変化で見る限り、Raw-エキソソームとリポソームは、凍結融解を30回繰り返すことにより60%程度脂質混合し、複合化が起こっていることが示唆された。
Raw exosome
Add 90 μL of 100 μM liposome solution to 10 μL of exosomes (150 μg / mL) co-stained with DIO and R18, solidify completely in liquid nitrogen, and repeat lysis 5 times in a 37 ° C heat block The sample was measured for fluorescence intensity and particle size. In addition, 1% of TRITONX-100 was added to the sample prepared in the same manner, and FRET was resolved with the fluorescence intensity at each number of times when the green fluorescence intensity was 100% under the condition that lipid mixing occurred completely. Efficiency 2 (Figure 4) was determined. As far as the change in fluorescence intensity is concerned, it was suggested that Raw-exosomes and liposomes were mixed with lipid by about 60% by repeating freeze-thawing 30 times, and complexing occurred.
また、エキソソームとリポソームを混合して凍結融解を30回繰り返した場合のみ、赤色の蛍光が増大した。R18は自己消光性の色素であり、その蛍光強度が増大したということはエキソソーム上のR18分子間距離が顕著に広がったことを示しており、このことからも複合化が起こっていることが示された。 In addition, red fluorescence increased only when exosomes and liposomes were mixed and freeze-thawed was repeated 30 times. R18 is a self-quenching dye, and its increased fluorescence intensity indicates that the distance between R18 molecules on the exosome has increased significantly, which indicates that complexation has occurred. It was done.
これまでの実験においては、脂質膜成分が混合することによりリポソームとエキソソームの複合化をモニターしていたが、エキソソームとリポソームの内容物の混合が測定できないかどうかをリポソームにRNA検出試薬であるGelStar(登録商標)を封入し、エキソソームと混合して凍結融解を行なって調べた。 In previous experiments, the complexation of liposomes and exosomes was monitored by mixing lipid membrane components. However, whether or not the mixing of the contents of exosomes and liposomes cannot be measured can be determined by using the RNA detection reagent GelStar. (Registered Trademark) was encapsulated, mixed with exosomes, freeze-thawed, and examined.
その結果、混合しただけの系と比較して、凍結融解によって1.5倍蛍光強度が高くなった。このことより、凍結融解によって外部の脂質の交換だけでなく、内容物の移行混合もおこっていることが示された。 As a result, the fluorescence intensity was increased 1.5 times by freezing and thawing as compared with the system in which only mixing was performed. From this, it was shown that not only the exchange of external lipids but also transfer mixing of contents occurred by freeze-thawing.
2. カチオン性リポソーム混合法
2.1実験手法
2.1.1. 試薬
DOPC [1,2-dioleoyl-sn-glycero-3-phosphocholine],
EPC [1,2-dioleoyl-sn-glycero-3-ethylphosphocholine]
DIO[3,3'-dioctadecyloxacarbocyanine perchlorate],
R18 [Octadecyl Rhodamine B Chloride],
2. Cationic liposome mixing method
2.1 Experimental method
2.1.1. Reagents
DOPC [1,2-dioleoyl-sn-glycero-3-phosphocholine],
EPC [1,2-dioleoyl-sn-glycero-3-ethylphosphocholine]
DIO [3,3'-dioctadecyloxacarbocyanine perchlorate],
R18 [Octadecyl Rhodamine B Chloride],
2.1.2. GL作製
クロロホルム:メタノール=2:1溶液に溶解したDOPCとEPCを目的比で混合し、DIOが1%になるように添加し、アルゴンガス環流下で溶媒を蒸発させて脂質フィルムを作成した。作成した脂質フィルムに100 μLの294 mMグルコース及び10 mM KCl溶液を加え室温で一晩静置し、GLを作成した。この時のDOPCとEPCの最終濃度が1 mMになるようにした。
2.1.2. Preparation of GL Lipid film by mixing DOPC and EPC dissolved in chloroform: methanol = 2: 1 solution at the desired ratio, adding DIO to 1%, and evaporating the solvent under argon gas reflux. It was created. GL was prepared by adding 100 μL of 294 mM glucose and 10 mM KCl solution to the prepared lipid film and allowing to stand at room temperature overnight. The final concentration of DOPC and EPC at this time was adjusted to 1 mM.
2.1.3. 細胞培養上清からのエキソソーム精製とエキソソームの染色
1.1.3. と同様に抽出したK562エキソソームを使用した。R18をDMSOに溶解し、最終濃度がエキソソームのリン脂質濃度の1%程度になるように加えた。そのとき、系に加わるDMSOの量は5%以下になるようにした。色素とエキソソームを混合した後、氷上で1時間静置し、カラムを用いて余剰色素を除去した。
2.1.3. Purification of exosomes from cell culture supernatant and staining of exosomes
K562 exosomes extracted as in 1.1.3. Were used. R18 was dissolved in DMSO and added so that the final concentration was about 1% of the exosome phospholipid concentration. At that time, the amount of DMSO added to the system was set to 5% or less. After mixing the dye and exosome, the mixture was allowed to stand on ice for 1 hour, and the excess dye was removed using a column.
2.2. 結果と考察
プレパラート上で緑色の蛍光色素であるDIOで染色したGL100 μM(lipid)溶液と赤色の色素であるR18で染色したエキソソーム溶液0.1 μg/μL(protein)を等量混合してレーザー共焦点顕微鏡[LSM]観察を行なった。その結果、時間とともに、リポソーム表面が厚くなることがわかり、カチオン性脂質であるEPC含有量が低い場合には、厚くなった部分が緑色で観察された(図7)。リポソームの表面にエキソソームが吸着したハイブリット体が得られた。
2.2. Results and discussion Laser prepared by mixing equal amounts of GL100 μM (lipid) solution stained with DIO, a green fluorescent dye, and exosome solution 0.1 μg / μL (protein) stained with R18, a red dye, on a preparation. The confocal microscope [LSM] observation was performed. As a result, it was found that the liposome surface became thicker with time, and when the EPC content, which is a cationic lipid, was low, the thickened portion was observed in green (FIG. 7). A hybrid in which exosomes were adsorbed on the surface of the liposome was obtained.
カチオン性脂質の含有量を70%まで増加した場合には、GLの表面が緑色の励起波長で赤色の蛍光が観察され、FRETが起こっていることがわかった。これは、GLとエキソソームの膜の複合化を示唆している。 When the cationic lipid content was increased to 70%, red fluorescence was observed at the green excitation wavelength of the GL surface, indicating that FRET occurred. This suggests a complex of GL and exosome membranes.
3. PEGによるリポソームの複合化とリポソームとエキソソームの相互作用
細胞やリポソームの複合化誘起剤として古典的に用いられているポリエチレングリコールを用いたリポソームとエキソソームの複合化を試みた。今回はPEG6000を用いて実験を行なった。
3. Complexation of liposomes with PEG and interaction between liposomes and exosomes We attempted to complex liposomes and exosomes using polyethylene glycol, which is classically used as a complexing inducer for cells and liposomes. This time, an experiment was conducted using PEG6000.
3.1実験手法
3.1.1. 試薬
DOPC [1,2-dioleoyl-sn-glycero-3-phosphocholine],
DOPS [1,2-dioleoyl-sn-glycero-3-phospho-L-serine]
DIO[3,3'-dioctadecyloxacarbocyanine perchlorate],
R18 [Octadecyl Rhodamine B Chloride],
3.1 Experimental method
3.1.1. Reagents
DOPC [1,2-dioleoyl-sn-glycero-3-phosphocholine],
DOPS [1,2-dioleoyl-sn-glycero-3-phospho-L-serine]
DIO [3,3'-dioctadecyloxacarbocyanine perchlorate],
R18 [Octadecyl Rhodamine B Chloride],
3.1.2. SUV作製
クロロホルム:メタノール=2:1溶液に溶解したDOPSに、NBD-PEとRhodamine-PEが1%になるように添加し、アルゴンガス環流下で溶媒を蒸発させて脂質フィルムを作成した。作成した脂質フィルムにPBSバッファーを150 μL加えて37℃で5分静置し、ボルテックスミキサーで撹拌した後エクストリューダーで100 nmのフィルターにかけてSUVを作成した。この時のDOPSの最終濃度が50 mMになるようにした。無色素のSUVも同様に作成し、コントロール実験に供した。
3.1.2. Preparation of SUV To DOPS dissolved in chloroform: methanol = 2: 1 solution, add NBD-PE and Rhodamine-PE to 1% and evaporate the solvent under reflux of argon gas to form a lipid film. Created. 150 μL of PBS buffer was added to the prepared lipid film, allowed to stand at 37 ° C. for 5 minutes, stirred with a vortex mixer, and then applied to a 100 nm filter with an extruder to prepare an SUV. The final concentration of DOPS at this time was adjusted to 50 mM. A dye-free SUV was prepared in the same manner and subjected to a control experiment.
3.1.3. 細胞培養上清からのエキソソーム精製
1.1.3. と同様に抽出したRawエキソソームを使用した。
3.1.3. Exosome purification from cell culture supernatant
Raw exosomes extracted as in 1.1.3. Were used.
3.1.4.エキソソームの染色と蛍光強度測定とナノサイト測定
2.1.3.と同様にDIOとR18で共染色したエキソソーム(150 μg/mL)10 μLに、PEGと脂質濃度にして90 μM分のDOPCsuvあるいはDOPSsuv溶液を加え、蛍光強度と粒子径の測定を行なった。
3.1.4. Exosome staining, fluorescence intensity measurement and nanosite measurement
As in 2.1.3, add 10 μL of exosomes (150 μg / mL) co-stained with DIO and R18 to a PEG and lipid concentration of 90 μM of DOPCsuv or DOPSsuv solution, and measure the fluorescence intensity and particle size. I did it.
3.2. 結果と考察
蛍光強度測定の結果、PEGの濃度が高くなるにしたがって緑色の蛍光強度が増加し、脂質成分の混合が起こっていることが分かった(図9, 10)。
3.2. Results and Discussion As a result of fluorescence intensity measurement, it was found that the green fluorescence intensity increased as the PEG concentration increased, and that lipid components were mixed (Figures 9 and 10).
また、ナノサイトによるサイズ測定の結果では、DOPCsuvよりも、DOPSsuvで、PEG
濃度が40%で顕著にサイズが大きくなり、複合化が生じていることが示唆された(図11)。
In addition, in the result of size measurement by nanosite, DOPSsuv, PEG
At 40% concentration, the size significantly increased, suggesting that complexation occurred (Figure 11).
本発明のベシクルは、薬物、核酸などの生理活性物質を細胞内に導入することができるので、ドラッグデリバリ−システム(DDS)として利用可能である。また、標識物質をベシクルに導入することで、バイオイメージングに応用することができる。さらに、抗原をベシクルに導入することで、ワクチンとして利用することもできる。 The vesicle of the present invention can be used as a drug delivery system (DDS) because physiologically active substances such as drugs and nucleic acids can be introduced into cells. In addition, by introducing a labeling substance into a vesicle, it can be applied to bioimaging. Furthermore, it can also be used as a vaccine by introducing an antigen into a vesicle.
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