JP7160385B2 - 脂質を用いた表面改質によって細胞内摂取効率を向上させたナノ粒子複合体及びその製造方法 - Google Patents
脂質を用いた表面改質によって細胞内摂取効率を向上させたナノ粒子複合体及びその製造方法 Download PDFInfo
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Description
1.ナノ粒子(NPs)の形成
アルブミン(Human serum albumin)を蒸留水に20mg/mLの濃度で溶かした後、0.2M NaOHを用いてpHを8に調節してアルブミン溶液を準備し、100%エタノールを1mL/minの速度で前記アルブミン溶液に滴定した。その後、4%のグルタルアルデヒド(glutaraldehyde)10μLを添加し、遮光下でエタノールを一晩蒸発させ、13200rpm、10minの条件で遠心分離を行った後、粒子化されていないアルブミンをピペットを用いて除去し、PBSで再分散して3000rpm、5minの条件で遠心分離を行った後、マイクロペレット(micropellet)を除く上澄み液(ナノ粒子(NPs))をピペットで得た(一方、蛍光実験を行う際には、必要による蛍光色素(dye)とナノ粒子とを一晩、常温で反応させた後、13200rpm、10minの条件で遠心分離を行い、反応していない蛍光色素(dye)をピペットを用いて除去した後、PBSで再分散して使用する)。
脂質DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine)とDSPE-PEG-NHS2000(1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol succinimidyl ester)を9.25:0.75のモル比で混合してクロロホルム(Chloroform)に10mg/mlの濃度で溶かし、1mg/mlとなるようにLCバイアル(vial)に100μlずつ仕込んだ後、窒素ガスを用いてクロロホルムを蒸発させ、しかる後に、デシケーター(desiccator)を用いて真空を作って1時間以上乾燥させて脂質薄膜(lipid film)を形成した。脂質薄膜バイアル(lipid film vial)にauto PBSを1ml仕込んで脂質溶液を形成し、55℃以上の温度を有する水に脂質溶液入りのHPLCバイアルを入れて脂質溶液の温度が55℃以上となるようにし、バスソニック(bathsonic)で15秒程度超音波処理(sonication)を実施することにより(このとき、熱水に浸す過程と超音波処理(sonication)実施過程を3回ほど繰り返し行う)、リポソームを形成した。
実施例1の2の過程を経て得られた結果物に、C3F8ガスを30秒間バイアル(vial)に満たし、バイアルミキサー(Vial Mixer)を用いて45秒間ミキシング(mixing)して、NHS反応基を含む脂質ベースのバブルを形成した。
(1)実施例1の1で形成されたナノ粒子をリポソーム溶液(実施例1の2で形成されたリポソームがPBSに1mg/mlの濃度で混合されて形成)に入れた後、NHS-アミン(NHS-amine)反応を誘導するために、RTで2時間以上反応させた後(このとき、リポソーム-ナノ粒子複合体(Liposome-NPs)が形成される)、超音波機器を用いて2W、1MHZ、デューティサイクル(duty cycle)100%の条件で5分以上機械力(mechanical force)を加えた。その後、破砕されたリポソームが脂質構造体を十分に形成することができるように、1時間以上RTでインキュベートして、ナノ粒子の外表面の一部分に脂質構造体が結合したナノ粒子複合体(Directional LT-NPs)を形成した。
1.実施例1の1で形成したナノ粒子(NPs)と実施例1の4の(2)で形成したナノ粒子複合体(Directional LT-NPs)をTEMで測定し、その結果を図2に示した。また、前記ナノ粒子複合体及び脂質構造体をCryo-TEMで測定し、その結果をそれぞれ図3および図4に示す。
1.実施例1の1で形成したナノ粒子(NPs)と実施例1の4の(2)で形成したナノ粒子複合体(Directional LT-NPs)に対する細胞摂取効率を評価するために、フローサイトメトリー(Flow cytometry)を用いて分析し、その結果を図5に示したとともに、共焦点顕微鏡を用いてイメージ化し、その結果を図6に示した。フローサイトメトリーを用いた分析は、A549細胞(1×104)にAlexa 488蛍光色素(dye)が標識されたナノ粒子及びナノ粒子複合体を処理して行った。共焦点顕微鏡を用いた分析は、DAPIを用いて核が染色され、ファロイジン(Phalloidin)を用いて細胞骨格(cytoskeleton)が染色されたA549細胞(1×105)に、Cy5.5蛍光色素(dye)が標識されたナノ粒子及びナノ粒子複合体を処理して行った。
1.エンドサイトーシス阻害剤(Endocytosis Inhibitor)を処理した細胞に対して、実施例1の1で形成したナノ粒子(NPs)と実施例1の4の(2)で形成したナノ粒子複合体(Directional LT-NPs)の細胞摂取効率を評価した。サイズ200nmのナノ粒子は、大きくマクロピノサイトーシス(macropinocytosis)、クラスリン非依存性エンドサイトーシス(clarthrin-independent endocytosis)、クラスリン依存性エンドサイトーシス(clarthrin-dependentend ocytosis)の合計3つのメカニズムで細胞内摂取が行われることが知られているので、これを阻害(inhibition)する阻害剤(inhibitor)を選定して、まず、A549細胞(1×104)をそれぞれまたは同時に1時間処理した後、Alexa488蛍光色素(dye)が標識されたナノ粒子及びナノ粒子複合体を3時間処理し、フローサイトメトリーを用いて測定した。測定結果について、ナノ粒子を基準に正常化(normalize)して、図8に示した。マクロピノサイトーシス(Macropinocytosis inhibitor)としては、EIPA(5-(N-Ethyl-N-isopropyl)amiloride)を選定(25μg/mlの濃度)してNa+/H+exchangeメカニズムを妨害し、クラスリン依存性エンドサイトーシス(Clathrin-dependent endocytosis inhibitor)としては、CPZ(chlorpromazine)選定(20μg/mlの濃度)してクラスリン被覆ピット形成(clathrin-coated pitformation)を妨害し、クラスリン非依存性エンドサイトーシス阻害剤(Clathrin-independent endocytosis inhibitor)としては、MβCD(methyl-β-cyclodextrin)を選定(3mg/mlの濃度)してクラスリン依存性エンドサイトーシスプロセス(cholesterol-dependent endocytic process)を妨害した。
1.アルブミン溶液にドキソルビシン(doxorubicin)が混合された溶液を添加して反応させた後、混合溶液が濁るまでエタノール滴定を行った以外は、実施例1の1及び実施例1の4の(2)と同様にして、ドキソルビシン含有ナノ粒子、ドキソルビシン含有ナノ粒子複合体を形成し、抗がん抵抗性を有する乳がん細胞株であるMCF-7/ADRをウェルプレート(wellplates)にシードし、それぞれドキソルビシン(100mM、DOX)、ドキソルビシン含有ナノ粒子(100mMのドキソルビシンを含む)及びドキソルビシン含有ナノ粒子複合体(100mMのドキソルビシンを含む)を含む培地中で37℃で6時間培養した後、前記細胞株をノーマル培地(normal media)中で48時間培養して細胞生存(Cell viability)を行うことにより、その結果を図9に示した。細胞生存(Cell viability)は、MTTアッセイとトリパンブルー色素排除法(trypan blue dye exclusion method)を用いるが、細胞生存(Cell viability)は、0.4%トリパンブルー色素(trypan blue dye)を有する細胞を培養し、ノイバウエル血球計(Neubauer hemocytometer)でカウントすることによって決定される。MTTアッセイで96ウェルプレートと1.5mg/ml MTT試薬(reagent)(3-(4,5-ジメチルチアゾール-2イル)-2,5-ジフェニルテトラゾリウムブロミド(3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide))を使用し、15μlのMTT試薬を持って2時間培養した後、200μlのDMSOを各ウェル(well)に追加し、Resulting culture platesをプレートリーダー(plate reader)(Bio Tek Instruments,Inc,Winooski,VT,USA)によって570nmで測定した。
1.アルブミン(Human serum albumin)を0.1mM HEPES with 0.01mM EDTAに40mg/mLの濃度で溶かした後、thiol-modify VEGF siRNA duplex(5’modified)を仕込み、アルブミンとsiRNAとの混合溶液が濁るまで100%エタノールを1mL/minの速度で滴定した後、遮光下でエタノールを一晩蒸発させ、13200rpm、10minの条件で遠心分離を行った後、粒子化されていないアルブミン或いは未反応薬物をピペットを用いて除去し、PBSで再分散して3000rpm、5minの条件で遠心分離を行った後、マイクロペレット(micropellet)を除く上澄み液(siRNAが担持されたナノ粒子(siRNA NPs))をピペットで得た。前記thiol-modify VEGF siRNA duplexのセンス(sense)は5’-AUGUGAAUGCAGACCAAAGAA-3’(配列番号:1)であり、アンチセンス(antisense)は5’-thiol-UUCUUUGGUCUGCAUUCACAU-3’(配列番号:2)である。
1.Tumor cell spheroid(腫瘍細胞スフェロイド)は、一般な接着性(adherent)がん細胞とは異なり、3次元培養(3D culture)を用いて増殖し、このようなスフェロイド(spheroid)は、がん細胞が培地上でフロート(floating)して成長するため、がん細胞がかたまって成長する。かたまって成長する形態ががん組織のECM(Extracellular matrix)をミミック(mimic)するという研究結果があるため、脂質表面の改質による組織透過(tissue penetration)有無をin vitroで確認するために、このモデルで実験を行った。
ポリ(2-ヒドロキシエチルメタアクリレート)(Poly(2-hydroxyethyl methacrylate))10gを100%純粋エタノール(pure ethanol)1Lに添加して60℃で溶かした後、溶かしたポリ(2-ヒドロキシエチルメタアクリレート)を100phi基準3.3ml程度プレート(plate)全体に均等に分散させて24時間乾燥させた後、コーティング(coating)を行い、用意されたプレートにMCF7細胞をシードし、5日間維持してスフェロイド(spheroid)を形成した細胞を得た。
(1)実施例1の1で形成したナノ粒子(NPs)と実施例1の4の(2)で形成したナノ粒子複合体(Directional LT-NPs)に対するスフェロイド(Spheroid)細胞摂取効率を評価するために、共焦点顕微鏡を用いてイメージ化し、その結果を図12に示した。DAPIを用いて核が染色された実施例7の2で形成されたスフェロイド(Spheroid)状のMCF-7細胞(1×105)に、Alexa555蛍光色素(dye)が標識されたナノ粒子及びナノ粒子複合体を処理した後、3時間が経過した時点で共焦点顕微鏡を用いて測定した。
2 脂質構造体
21 脂質ヘッド
22 脂質尾
Claims (2)
- 第1反応基が存在するアルブミンナノ粒子を形成するナノ粒子形成ステップと、
前記第1反応基と化学的に結合する第2反応基が存在するリン脂質ベースのマイクロサイズの脂質体を形成する脂質体形成ステップと、
前記ナノ粒子と脂質体とを混合して第1反応基と第2反応基とが互いに結合するようにして、脂質体の外面にナノ粒子が結合した脂質体-ナノ粒子複合体を形成する脂質複合体形成ステップと、
前記脂質複合体形成ステップで形成された脂質体-ナノ粒子複合体に機械力を加えて脂質体を破砕することにより、ナノ粒子の外表面の一部分に結合したチューブ形状の脂質構造体を形成してナノ粒子複合体を製造する破砕形成ステップと、を含み、
前記脂質体は、バブルまたはリポソームであり、
前記ナノ粒子は、細胞内に摂取されて疾病の治療に用いられ、前記脂質構造体は、ナノ粒子の細胞内摂取効率を向上させることを特徴とする、ナノ粒子複合体の製造方法。 - 前記破砕形成ステップでは、脂質体-ナノ粒子複合体に機械力を加えて一定時間維持することにより、脂質体が破砕され、脂質体をなすリン脂質は組み換えが起こり、ナノ粒子に結合したチューブ形状の脂質構造体が形成されることを特徴とする、請求項1に記載のナノ粒子複合体の製造方法。
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