JP6786518B2 - カルボシランデンドリマーを用いた薬物送達システム用標的型シェル - Google Patents
カルボシランデンドリマーを用いた薬物送達システム用標的型シェル Download PDFInfo
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- JP6786518B2 JP6786518B2 JP2017554152A JP2017554152A JP6786518B2 JP 6786518 B2 JP6786518 B2 JP 6786518B2 JP 2017554152 A JP2017554152 A JP 2017554152A JP 2017554152 A JP2017554152 A JP 2017554152A JP 6786518 B2 JP6786518 B2 JP 6786518B2
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Description
さらに、蛍光タンパク質を担持させたシロール含有カルボシランデンドリマーによって形成されるミセルの内径は、およそ50nm〜500nm程度であること、及びこのような内径を有するミセルには、その内部に生体高分子を含む種々の薬剤を内包させることが可能であることをも見出した。
DMPGTVLPGG(配列表の配列番号1)
VPTDTDYSGG(配列表の配列番号2)
DMPGTVLPGG GGGSEGEWQ QQQHQWAKQE(配列表の配列番号3)
本発明のカルボシランデンドリマーは図2に示す手順(スキーム1)で合成することができる。
(実施例1)DDS用凝集性分子及びミセルの作製
本実施例においては、標的提示部作製用のタンパク質として下記の通りのGFPを、また、デンドリマーとしてシロールデンドリマーを用いた。
(1)DDS用凝集性分子の作製
本実施例において、ハロゲン基を有するデンドリマーとして、下記の化学式(X)に示す化合物(以下、「ジメチルダンベル(1)6−Br」ということがある。)を使用し、チオール基を有するタンパク質として、GFP(緑色蛍光タンパク質)(配列番号7)を用いた。
本実施例において使用したシロールデンドリマーは、疎水性のシロールのコア部分が凝集すると発光する(AIE効果を発揮する)性質を有しているため、ミセル構造をとった場合にも発光することが確認されていた。そこで、シロールデンドリマーにGFPを結合させたGFP-シロールデンドリマーからなるミセルを作製した場合に、シロールとGFPとの間において蛍光共鳴エネルギー移動(FRET:Fluorescence resonance energy transfer)が生じるがどうかを検討した。
以上から、本発明のミセルをDDS用凝集性担体として使用すると、送達された組織・器官を確認できることが示された。また、標的組織・器官に送達されたミセルが崩壊した後であっても、蛍光タンパク質の蛍光を追跡することはでき、これによって、蛍光タンパク質が送達された細胞内の環境等を感知することもできる。
次に、本発明のミセルを用いて、薬剤等を内包するミセルが作製できるか否かを確認した。本実施例では、モデル薬剤として、DiI(1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate、Promokine PK-CA707-60010, PromoCell GmbH製)、Oil orange SS(東京化成工業(株)製、T0553)、ヤギ抗マウスIgG(abcam社製、ab6708)-Alexa610(Molecular Probes社製、A30050)およびWGA(Wheat Germ Agglutinin:小麦胚芽レクチン、Molecular Probes社製)(WGA-Alexa Fluoro (登録商標) 594 conjugate、W11262)を用いた。
本願発明のミセルとC末端で結合し、かつ、癌細胞の表面に発現する受容体と結合する標的ペプチドとN末端で結合する、標的ペプチド配列結合蛍光タンパク質を以下のように調製した。
(1−1)ペプチド配列の選定
選定した標的結合部位に組み込んだペプチドのアミノ酸配列を下記表3に記載した。MCF7-1は、下記表3に示す配列を含む、ヒト乳腺癌由来細胞であるMCF7であり(以下、「標的タイプ1型」ということがある。)、MCF-2は、下記表3に示す配列を含むMCF-1の変異体(以下、「標的タイプ2型」ということがある。)である。また、MCF7-1+αスタンドは、下記表3に示すように、上記のMCF7-1にαへリックス構造の短いペプチド(以下、「αスタンド」という。)をつないだ構造を有するMCF7-1の別の変異体である(以下、「タイプ1強化型」ということがある。)。
表3に示された上記ペプチド配列についてインバースPCRを行なうためのプライマーを下記表4に記載した。これらプライマーは、配列表の配列番号1及び3については上記ペプチド配列のDMとPGTVLPの間から、配列番号2については上記ペプチド配列のVPとTDTDYSGGの間から伸長反応が開始するように設計した。これはインバースPCRが最適に行われることを鑑みている。
インバースPCR用テンプレートプラスミドは、以下の論文に記載の方法で調製した。
「Protease-sensitive signaling by chemically engineered intramolecular fluorescent resonance energy transfer mutants of green fluorescent protein. - Miho Suzuki, et al. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression
Volume 1679, Issue 3, 17 September 2004, Pages 222-229」
GFPuv5は、pGFPgcn4から以下のようにして得た。まず、I167T突然変異+フォワードプライマー 5’CATTGAAGATGGCTCCGTTCAA(配列番号18)及びリバースプライマー5’TTGTGGCGAGTTTTGAAGTTAG(配列番号19)を用いたインバースPCRを用いて遺伝子マニピュレーション用の同義の突然変異を生じさせたPCR産物を得て、引き続きこのPCR産物を環化処理して作製された。以上のようにして得られたコンストラクトを、pGFPgcn5と命名した。
次に、システインを6位及び229位にそれぞれインバースPCRによって導入した。この導入には、C48S突然変異を有するプラスミドと、以下のプライマーのセットをそれぞれの突然変異に使用した。
Ile置換用:5’TGCACACATGGCATGGATGAGCTC(配列番号28)及び5’CCCAGCAGCAGTTACAAACTC(配列番号29)
トリプシン標的配列(Gln-Gly-Arg)からなる3つのプロテアーゼタグは、種々のスペーサー配列を有し(スペーサーなし、Thrスペーサー又はGly-Thrスペーサー)、必要なシステインがHis-231とAsp-234との間で置換されている。このコンストラクトを、puvC48Stagと得られたプラスミド(テンプレート)と、下記表5に示すプライマーとを用いて得た。
全てのプラスミドで大腸菌BL 21(DE3)を形質転換した。アンピシリン(50μg/ml)及びIPTG (0.5mM)を添加したLB培地(38mL)に、定常期の終夜培養した大腸菌(12ml)を播種し、37℃で8時間インキュベートした。2,500 x gで20分間遠心して細胞を集め、10mLのPBSに再懸濁した。細胞のペレットを、50mMトリス及び8M尿素を含む10mLの溶解バッファー(pH 8.0)中で、15分間、室温にて溶解させ、ボルテックスした。1,200 x gで15分間遠心し、上清をPBS中に懸濁したNi2+-NTA 樹脂(Qiagen Co. Ltd.製)と混合した。この樹脂を、PBS及び20mMのイミダゾールで順番に洗浄した後に、結合したGFPuv5tag突然変異体を250mMのイミダゾール溶液で溶出させた。
インバースPCR用テンプレートプラスミドの塩基配列を配列番号26に示した。
下記表6に示す反応液を調製し、下記表7の条件でインバースPCRを行った。
上記PCR反応液の一部をとって、0.8% PAGE、電圧100 V、印圧時間30分間のゲル電気泳動を行い、各サンプル中で増幅されたペプチドを確認した。泳動結果を図9に示す。
下記表8の反応液を調製し、120℃で30分間反応させ、上記PCRによって生じた非依存的A配列を除去した。その後、QIAquick(登録商標) PCR Purification Kit(QIAGEN社製)を使用して、キットに添付の説明書に従って上記PCR産物を精製した。
引き続き、下記表9に示す反応液を調製し、16℃で3時間以上反応させて、後述する大腸菌DH5αの形質転換用環状プラスミドを作製した。変異体に応じて、25℃、37℃等を使い分けた。
大腸菌DH5αのコンピテントセル(BioDynamics Laboratory社製)10μlを使用直前に氷上で融解させ、コンピテントセル溶液を調製した。このコンピテントセル溶液にライゲーション反応液を1μL加え、氷上で30分間放置した。その後、42℃で30秒間インキュベートした後に、氷上で2分間冷却した。ここに、SOC(東洋紡(株)製)培地を90μL加え、37℃で1時間シェーカー上において反応させた。その後、アンピシリン含有LB選択培地(東洋紡(株)製)に播種し、37℃で一晩静置した。
上記形質転換で得られたコロニーからコロニーPCRを行い、想定されるインサートを確認した。
(5−1)PCR用反応液溶液の調製
下記表10のコロニーPCR用反応液を調製した。
上記PCR反応液の1.2%アガロースゲルで印加電圧100 V、時間30分間の電気泳動を行い、増幅が確認できたコロニーを、LB液体培地(東洋紡(株)製)が入った培養ボトルに移植し、37℃でインキュベートした。
上記LB液体培地で培養した大腸菌中の上記プラスミドを、Wizard Plus SV Minipreps. DNA Purification System(プロメガ(株)製)を用い、製品に添付された説明書に従って精製した。その後、精製したプラスミドの配列をユーロフィンジェノミクス(株)に依頼して解析した。
大腸菌BL21(DE3)コンピテントセル(BioDynamics Laboratory社製)10μLを使用直前に氷上で融解させ、コンピテントセル溶液を調製した。このコンピテントセル溶液に、上記シーケンスにより目的とする配列が入っていることが確認できたプラスミド液1μlを加え、氷上で30分間放置した。
(8−1)コロニーの培養
50mLチューブにアンピシリン含有LB液体培地4mLを入れ、ここに上記前培養液290μLを加えたものを試料として4本用意し、28℃で4時間シェーカー上で培養した。その後、100mMのIPTG(イソプロピル-β-チオガラクトピラノシド)を43μL加え、28℃で一晩シェーカー上で培養した。
翌日、上記の4本のチューブの培養液を1本のチューブに集約した。内容物を移した3本のチューブを、それぞれ1mlのPBS(-)バッファー(和光純薬工業(株)製)で洗浄し、この洗浄液もまとめたチューブに加えた。以上のように培養物を集約したチューブを、室温にて、5,000rpm(遠心機名:KUBOTA3740、ローター番号KUBOTA AF2018、久保田商事(株)製)で5分間遠心した。
Ni-NTA Agarose(QlAGEN社製)を15mLチューブに2mLとり、(i)1,000rpmで1分間遠心し、(ii)上清を捨てて、1×PBSバッファーを1mL加えてよく攪拌した。上記(i)と(ii)とを3回繰り返し、Ni-NTA樹脂を調製した。
上記タンパク質精製溶液中のイミダゾールとPBSバッファーとを置換し、精製して目的の標的ペプチド配列結合蛍光タンパク質を得た。以上の操作では、GEヘルスケア・ジャパン株式会社のNAP5カラムを使用し、製品に添付の説明書に従って精製を行った。
上記精製作業で得られた標的ペプチド配列結合蛍光タンパク質濃度の吸光度(280nm)、及び発色団濃度(形成能)の吸光度(488nm)を常法に従って計測し、A488/A280の値が1.5を超えたものを、目的の標的ペプチド配列結合蛍光タンパク質として選択した。結果を表12に示した。
タンパク質としてGFPに代えて、実施例2で調製した標的ペプチド配列結合蛍光タンパク質を用いて、実施例1と同様にして、標的ペプチド配列が結合したミセル(蛍光タンパク質会合駆動型ミセル)を作成した。
上記作製した標的ペプチド配列結合蛍光タンパク質会合駆動型ミセルの発光特性を、実施例1と同様にして測定した(図10参照)。図10の凡例中、反応物は、シロールデンドリマーと標的ペプチド配列結合蛍光タンパク質が結合した複合体を、未反応物は、それらが結合していない状態のものを示し、各数値は励起光の波長(nm)を示す。標的ペプチド配列結合ミセルでも、実施例1と同様に、510nm付近に発光のピークが観察された。
上記標的ペプチド配列結合リポソーム又はミセルの粒子特性を、実施例1と同様に動的光散乱法により測定した(図11参照)。図11中、横軸は得られたミセルの粒径を、縦軸は各横軸に示すサイズのミセルの全体に占める%を表す。その結果、実施例1と同様に、およそ100nm〜200nmの粒子径を有する粒子が確認された。
以上から、標的ペプチド配列を有する蛍光タンパク質結合ミセルも、標的配列を有しないそれと同等のミセルを形成することが示された。さらに、実施例1で得られたミセルも、本実施例で得られたミセルのいずれもが、蛍光タンパク質の会合によって形成される、蛍光タンパク質会合駆動型ミセルであると推定された。
(1)基本リポソーム又はミセルの作製
リポソーム又はミセルを以下のようにして作製した。まず、GFP含有溶液を、10K アミコンフィルタ(Amicon社製)を用いて、14,000 x gで15分間遠心して濃縮し、1 x PBSを加えて99μLにメスアップした。20μMのGFP含有溶液を使用して、50μM/50μLのミセルを作製するために、137.5μLのGFPを濃縮した。
次いで、終夜で凝集させた約380μLの溶液全量を100 Kアミコンフィルタに入れ、14,000 x gで10分間遠心して濃縮し、約30μLをカラム上液として取った。ここに100μLの1 x PBSを加えて14,000 x gで10分間遠心して洗浄した。この洗浄操作を3回繰り返し、洗浄液約300μLを集めた。
低温低真空走査型電子顕微鏡((株)日立ハイテクノロジーズ製、カタログ番号 S-3400N)を用いて、リポソーム又はミセルの判別を行ったところ、概ね観察された分子はリポソーム(ベジクル)であった。液体窒素雰囲気下で切片の観察を行なった。
(1)試料ミセル(標的結合部位を有するミセル)の作製
50μM x 50μL分の試料ミセルを、以下のようにして作製した。基本ミセルとして、上記実施例(X1)で作製した未標的型ミセル(NSS25及びNSS26)を、それぞれ16.1μM及び31.6μM使用した。標的認識部位としてMCF7の配列を含む標的型ミセル(配列表の配列番号12及び13)を11.3μM使用した。
ついで、各リポソーム又はミセル溶液をNICKカラムで処理し、得られた各処理液にTPSを3.21μL加え、37℃で終夜置いた。
上記(1)のようにして得た各ミセル溶液を、それぞれ380μLずつAmicon 100Kフィルタに入れ、14,000 x g で10分間遠心した。約350μLがフィルタを抜けて落ちるため、これを下液、フィルタ上に残った約30μLを上液とした。
(1)タンパク質の精製
上記のミセルの構成タンパク質の物性を確認するために、Ni NTA Super flow(Qiagen社製)を用いて上記構成タンパク質を以下の手順で精製した。
まず、基本ミセル(50μM/50μL)を50μL取って50mLのファルコンチューブ(ファルコン社製)に入れ、20mMのイミダゾールで洗浄し、その後、250mMのイミダゾールを加えた。回転シェーカーで終夜反応させ、ミセルからタンパク質を溶出させた。このファルコンチューブをそのまま遠心機(久保田商事(株)製)にセットして、12,000rpmで10分間遠心し、得られた上清を15mLのファルコンチューブに移した。
上記のようにファルコンチューブを遠心している間に、Ni NTA樹脂(Ni NTA Super flow中に含まれている)の準備を行なった。まず、Ni NTAアガロースを2.5mL取り、防腐剤を除去した。次いで、Ni NTAアガロースゲルを含むこの溶液を、よく振って均一に混合し、15mLのファルコンチューブに移した。
15mLのファルコンチューブ中の上記の構成タンパク質溶液中に、上記スラリーがチューブの内壁につかないように注意しながら、400μLを滴下した。その後、回転シェーカーにセットして最速にメモリを合わせて10分間反応させた。
以上のようにして精製した各タンパク質(GFP)についてゲル電気泳動を以下のようにして行った。
まず、5μMの各タンパク質を含む溶液を約10μLずつ準備し、1.5mLの蓋付きチューブに入れた。分子量マーカー(ラダー)として、Precision Protein Standards Prestained Broad Range(BioRad社製)も10μLを取って、同様に1.5mLチューブに入れた。下記表13に示す組成のランニングゲル(12%)及び表14に示す組成のスタッキングゲル(4%)を調製した。
内包させる薬剤として、Orange OTを使用した。80μMのGFP含有溶液を使用し、7.78mMのTPSを10.28μL炭化した以外は、上記基本ミセルの作製工程と同じ手順でミセル溶液を調製した。ついで、Orenge OT(8mMのOrange OTのストック溶液)を、GFPに対して、モル比(終濃度)で1:1となるよう1μL加えた。次いで、DiI282(400μMのDiI282のストック溶液)を、GFPに対して、モル比(終濃度)で20:1になるよう加え、37℃にて終夜で取り込ませた。
上液については、基本リポソーム又はミセルの作製と同じ手順で処理し、同じ希釈倍率で、粒度及び蛍光測定を行った。薬剤の内包は疎水性、親水性物質ともに可能と思われるがミセル形成条件の最適化が各々必要であると考えられた。
(1)実験方法
コンフレントになっているMCS7細胞を、0.25%のtrypsin溶液にて剥がし、細胞懸濁液(1 x 109個/mL)を調製した。この細胞懸濁液を、3mLずつ、5枚のコラーゲンコードディッシュ(MatTek社製)又はポリ-d-リジンディッシュ(MatTek社製)に播種した。
DAPIについては405nmのレーザーで励起し、460nmの発光を観察して、デンドリマーの部分を観察した。また、GFP部分は515〜520nmの発光を観察した。観察結果を図20A〜20Cに示す。3時間の結果(図20C上段)と24時間の観察結果(図20C下段)とを比較したところ、24時間でも、標的性を持たせたミセルの方が、標的細胞に正しく取り込まれているように思われた(図16〜18参照)。
解析ソフトにはImage Jを用いた。それぞれのエリアの蛍光強度を数値化し、ミセルの蛍光強度と蛍光タンパク質の蛍光強度の比率より、ミセルの崩壊、蛍光タンパク質の残存を確認するに至った。
配列番号2:GFPに組み込む標的認識配列ペプチド(MCF7-2)
配列番号3:GFPに組み込む標的認識配列ペプチド(MCF7-1+αスタンド)
配列番号4:MCF7-1を組み込んだGFP
配列番号5:MCF7-2を組み込んだGFP
配列番号6:MCF7-1+αスタンドを組み込んだGFP
配列番号7:GFPのアミノ酸配列
配列番号8:GFPのアミノ酸配列
配列番号9:BFPのアミノ酸配列
配列番号10:YFPのアミノ酸配列
配列番号11:イソギンチャクモドキ由来の蛍光タンパク質のアミノ酸配列
配列番号12:MCF7−1の増幅用フォワードプライマー
配列番号13:MCF7−1の増幅用リバースプライマー
配列番号14:MCF7−2増幅用フォワードプライマー
配列番号15:MCF7−2増幅用リバースプライマー
配列番号16:MCF7-1+αスタンド増幅用フォワードプライマー
配列番号17:MCF7-1+αスタンド増幅用リバースプライマー
配列番号18:インバースPCR用フォワードプライマー
配列番号19:インバースPCR用リバースプライマー
配列番号20:インバースPCR用フォワードプライマー
配列番号21:インバースPCR用リバースプライマー
配列番号22:オリゴヌクレオチド
配列番号23:オリゴヌクレオチド
配列番号24:オリゴヌクレオチド
配列番号25:オリゴヌクレオチド
配列番号26:インバースPCR用フォワードプライマー
配列番号27:インバースPCR用リバースプライマー
配列番号28:インバースPCR用フォワードプライマー
配列番号29:インバースPCR用リバースプライマー
配列番号30:インバースPCR用フォワードプライマー
配列番号31:インバースPCR用リバースプライマー
配列番号32:インバースPCR用フォワードプライマー
配列番号33:インバースPCR用リバースプライマー
配列番号34:インバースPCR用フォワードプライマー
Claims (8)
- 水性溶媒中で前記凝集性分子が凝集して形成され、50〜500nmの直径を有する、請求項1に記載のドラッグデリバリーシステム用標的型シェル。
- 前記ペプチドは、前記標的組織に発現されている、表面抗原、受容体、ゲート、トランスポーター及びチャンネルからなる群から選ばれるいずれかの標的タンパク質と特異的に結合し、前記標的タンパク質と前記ペプチドの結合体が細胞内へのエンドサイトーシスを促進するものであることを特徴とする、請求項1に記載のドラッグデリバリーシステム用標的型シェル。
- 前記標的組織は、炎症を起こしている正常組織、好ましくない遺伝子発現の見られる細胞を含む組織、好ましくない遺伝子発現の見られる細胞で構成される組織、及び腫瘍細胞で構成された組織からなる群から選ばれるいずれかの組織であることを特徴とする、請求項1に記載のドラッグデリバリーシステム用標的型シェル。
- 前記蛍光タンパク質は、赤色蛍光タンパク質、黄色蛍光タンパク質、青色蛍光タンパク質、及び緑色蛍光タンパク質からなる群から選ばれるいずれかの蛍光タンパク質であることを特徴とする、請求項8に記載のドラッグデリバリーシステム用標的型シェル。
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PCT/JP2015/083622 WO2016084979A1 (ja) | 2014-11-28 | 2015-11-30 | カルボシランデンドリマー、そのデンドリマーを用いた薬物送達システム用凝集性担体 |
JPPCT/JP2015/083622 | 2015-11-30 | ||
JP2016106700 | 2016-05-27 | ||
JP2016106700 | 2016-05-27 | ||
PCT/JP2016/085604 WO2017094792A1 (ja) | 2015-11-30 | 2016-11-30 | カルボシランデンドリマーを用いた薬物送達システム用標的型シェル |
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WO2020132875A1 (zh) * | 2018-12-25 | 2020-07-02 | 深圳迈瑞生物医疗电子股份有限公司 | 红细胞模拟粒子、其制备方法及含其的质控物或校准物 |
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CN102781965A (zh) | 2009-10-06 | 2012-11-14 | 安吉奥开米公司 | 用于转运治疗剂的组合物和方法 |
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