JP2018031763A - 抗体および磁性ナノ粒子が結合された導電性高分子を含む血中のがん細胞の検出および回収用の磁性ナノ構造体 - Google Patents
抗体および磁性ナノ粒子が結合された導電性高分子を含む血中のがん細胞の検出および回収用の磁性ナノ構造体 Download PDFInfo
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Abstract
Description
しかし、今までがんの診断方法は組織サンプルの採収および内視鏡の検査などの侵襲的な方法が主になっており、従来の侵襲的な診断および検査方法の代案としてリキッドバイオプシーが注目されている。リキッドバイオプシーは血液などの体液検査のみで身体部位別の血液内に存在するがん細胞由来のDNAを解析してがんの発生および転移などに関する詳しい観察ができ、非侵襲的な(non−invasive)方法を用いるため、遺伝体解析技術の急速な発展とコストの節減のメリットに基づいてがん診断技術へ応用しようとする努力が試みられている。
これで、従来の血中のがん細胞の検出技術の問題点を解決して検出効率を高めることができる技術の開発が求められているのが実情である。
また、本発明は本発明によるナノ構造体から検出した血中のがん細胞からDNAを抽出または分離して解析するステップを含むがんの発病および/または予後を診断するための情報提供方法を提供する。
また、本発明は本発明のナノ構造体を含む血中のがん細胞の検出および回収用の組成物を提供する。
以下、本発明の理解を助けるために望ましい実施例を提示する。しかし、下記の実施例は本発明をより容易に理解するために提供されているものであり、下記の実施例により本発明の内容が限定されるものではない。
1−1.抗体混合物が結合されたポリピロール磁性ナノ粒子(Ab mixture_mPpyNPs)の製造
ヒアルロン酸(Hyaluronic acid)が結合されたポリピロール(Ppy)ナノ粒子の合成のために、超純水(ultrapure water)3.125mlにポリビニルピロリドン(Polyvinyl pyrrolidone;PVP、M.W:29,000)0.125gを溶解させた後、30分の間、室温で強く攪拌した。その後、16.25mlのピロール(pyrrole)および1.5mlの10nmの磁性ナノ粒子を柔らかく攪拌しながら添加した。10分後、125μlの塩化鉄(II)六水和物(iron(II) chloride hexahydrate、0.75g/ml)および100mgのヒアルロン酸(40K)を迅速に添加して、常温で3時間、重合させた。生成物は2日間透析により精製した後、精製した溶液を凍結乾燥して、使用するまでに真空状態で保管した。その後、約2mgのヒアルロン酸が結合されたポリピロールナノ粒子(HA−Ppy NPs)を1mlの0.4M EDC(N−(3−Dimethylaminopropyl)−N′−ethylcarbodiimide hydrochloride)および0.1M NHS(N−hydroxy succinimide)で45分間、混合した後、17,000rpmで遠心分離を行った。最後に、10μl/mlの濃度で混合されたビオチンが付着された抗体(EpCAM、EGFR、N−cadherin、TROP−2およびvimentin)と結合する10μl/mlのストレプトアビジン1mlにヒアルロン酸が結合されたポリピロールナノ粒子(HA−Ppy NPs)を再懸濁した。製造した溶液を再度17,000rpmで遠心分離して、使用するまでに超純水(ultrapure water)に保管した。
多孔性アルミナ鋳型(AAO template、Whatman、pore diameter、200nm)の片面に約150nmの厚さの金(Au)層を加熱蒸発(thermal evaporation)させ蒸着させた。すべての電気化学的な実験は金(Au)でコーティングされたAAO鋳型で白金ワイヤの相対電極とAg/AgCl(3.0M NaCl type)の比較電極を具備したpotentiostat/galvanostat(BioLogic SP−150)を用いて測定した。図1bに図式するように、抗体混合物が結合されたポリピロール磁性ナノワイヤ(Ab mixture_mPpyNWs)の製造のために、常温で30μlの磁性ナノ粒子(−10nm in a diameter)を金(Au)でコーティングされたAAOディスクの上に付着させ、AAOの細孔(pore)に導入した。磁性ナノ粒子が高い密度でドーピング(doping)された、五つの類型の抗体が結合されたポリピロール(Ppy)ナノワイヤの製造のために、AAO鋳型の細孔に0.01Mポリ(4−スチレンスルホン酸)(poly(4−styrene sulfonic acid))および1mg/ml NHS−SS−biotin(Succinimidyl−2−(biotinamido)−ethyl−1,3′−dithiopropionate)を含有する0.01Mピロール溶液と一緒に1.0V(vs.Ag/AgCl)で7分間クロノアンペロメトリー(chronoamperometry)を適用して電気化学的な蒸着を行った。その後、磁性ナノ粒子およびSS−biotin分子がドーピングされたフリースタンディングポリピロールナノワイヤ(free―standing Ppy NWs)を得るためにAAO鋳型を超純水(ultrapure water)で数回洗浄して、2Mの水酸化ナトリウム(NaOH)溶液に浸した。続いて、ポリピロールナノワイヤ(Ppy NWs)のカルボン酸(−COOH;carboxylic acid)基を活性化するためにポリピロールナノワイヤ(Ppy NWs)に30mMのEDCおよび6mMのNHS(N−hydroxy succinimide)を添加した。生成されたポリピロールナノワイヤ(Ppy NWs)をストレプトアビジン(10μg/ml)と一緒に追加で45分間培養した後、水で洗浄した。続いて、ビオチンが付着された抗体混合物(つまり、biotinylate anti−EpCAM、biotinylated anti−EGFR、biotinylated anti−N−cadherin、biotinylated anti−TROP−2およびbiotinylated anti−vimentin、(10μg/ml in PBS))をポリピロールナノワイヤ(Ppy NWs)の末端にストレプトアビジンが導入されたポリピロールナノワイヤ(Ppy NWs)と一緒に4℃で一晩反応させ、抗体混合物が結合されたポリピロール磁性ナノワイヤ(Ab mixture_mPpyNW)を製造した。抗体混合物が結合されたポリピロール磁性ナノワイヤ(Ab mixture_mPpyNW)の形態(morphology)は加速電圧15kVの走査電子顕微鏡(G2F30、Tecnai)および300kVの透過電子顕微鏡を用いて観察した。磁気力の測定はSQUID−VSM magnetometer(MPMS−VSM、Quantum design)を用いて常温で行った。磁場は70ないし70kOeの強さで変化させ印加して、横緩和時間(transverse relaxation time、T2)は7テスラ(Tesla)MRI(Bruker BioSpin MRI GmbH;echo time[TE]=6.5msおよびrepetition time[TR]=1,600ms)を用いて測定した。
2−1.単一抗体または抗体混合物が結合された磁性ナノワイヤの細胞捕集効率の比較
EpCAM−陽性細胞(HCT116大腸がん細胞、MCF7乳がん細胞)およびEpCAM−陰性細胞(MDA−MB−231乳がん細胞、MIA PaCa−2膵臓がん細胞)はAmerican Type Culture Collection(ATCC)から購入して、DMEMまたは10%のウシ胎児血清(FBS)が添加されたRPMI−1640に100unit/mlのペニシリン/ストレプトマイシンが補充された培地に、37℃で5%CO2で加湿された培養器で培養された。細胞培養試薬はThermo Scientific Hyclone and Gibcoで購入した。抗体混合物が結合されたポリピロール磁性ナノワイヤ(Ab mixture_mPpyNWs)および単一抗体(EpCAM抗体)が結合されたポリピロール磁性ナノワイヤ(EpCAM_mPpyNWs)の細胞捕集効率を評価するために、上記の実施例1−2に記載された方法で抗体が結合されたポリピロール磁性ナノワイヤを製造した。EpCAM−陽性細胞(HCT−116、MCF7)およびEpCAM−陰性細胞(MIA PaCa−2、MDA−MB−231)を含む四つの相違な細胞を、0.1%PBS(phosphate buffer saline)/BSA(bovine serum albumin)または健康な供与者から得た全血に100cells/mlの濃度で処理した後、30分間常温で、細胞懸濁液に磁性ナノワイヤを添加して、標的細胞が捕集されるように柔らかく攪拌させた後、培養した。その後、磁石により生成された磁場を用いて捕集された細胞を効率的に分離するために、1.5mlのマイクロ遠心分離チューブを用いて、捕集された細胞を分離した。磁石を用いた分離はサマリウムおよびコバルト磁石(samarium/cobalt magnet)を含むMagneSphere(登録商標) Technology Magnetic Separation Stands(Promega、USA)で行って、おおよそ128ないし264kJ/m3に該当する16ないし33megagauss−oersteds(MGOe)エネルギ(BHmax)範囲で細胞の分離を行った。
最適の細胞捕集効率をみせる磁性ナノワイヤの濃度を確認するために、上記の実施例1−2の方法で製造した抗体混合物が結合されたポリピロール磁性ナノワイヤ(Ab mixture_mPpyNWs)を用いて、HCT116細胞を20cells/mlの濃度で0.1%BSA/PBSに処理した後、磁性ナノワイヤの濃度を異なるようにして細胞の捕集効率を確認した。
磁性ナノ構造体および細胞の直接的な相互作用を通して、捕集性能を比較するために、上記の実施例2−1の方法で細胞を処理して、二つの異なる細胞株(Ep−CAM−陽性 HCT116細胞およびEpCAM−陰性 MDA−MB−231細胞)を用いて抗体混合物が結合されたポリピロール磁性ナノワイヤ(Ab mixture_mPpyNWs)および抗体混合物が結合されたポリピロール磁性ナノワイヤ(Ab mixture_mPpyNPs)の捕集効率を比較した。
3−1.捕集された細胞の免疫蛍光染色の確認
乳がん患者の抹消血液から稀な血中循環腫瘍細胞(CTC)を分離して、ポリピロールナノワイヤ構造体の性能を評価するために、総29人のがん患者の血液を検査した。全血は国立がんセンター(NCC)臨床試験審査委員会の承認手続きによりanti−coagulant EDTAと一緒に真空採血器のチューブに収集した。臨床適用のために、18人の健康な志願者および初期段階の乳がん患者29人から血液サンプルを収集した。ほとんどの患者は局所的な初期の乳がん(段階IおよびII)であって、29人の中に6人は手術の前に補助抗がん化学療法を受けた。別に処理されていない患者の血液250μl−1mlのサンプルを用いてCTC分離および解析を行った。また、対照群で健康な寄贈者の抹消血液250μl−1mlを用いてCTC検出を評価した。患者のサンプルで捕獲した細胞は周りの白血球と仕分けるために、DAPI、anti−EpCAM、anti−CD44、anti−vimentinおよびanti−CD45の抗体を用いて免疫組織学的な解析を通して確認した。
上記の実施例2−1の方法で細胞を捕集した後、捕集された細胞の免疫組織化学の解析を行うために、免疫化学染色を実施した。捕集された細胞は上皮マーカーであるEpCAMおよびジアミノベンジジン(diaminobenzidine;DAB)で染色して、ヘマトキシリン(hematoxylin)で対照染色した。また、SuperPicture 3rd Gen IHC検出キット(Invitrogen)を製造社のプロトコルに従い使用して、捕集された細胞はglass slideで400×の倍率の顕微鏡(Olympus BX52 microscope)およびAperio ImageScopeイメージ解析プログラムを用いて行った。
抗体混合物が結合されたポリピロール磁性ナノワイヤ(Ab mixture_mPpyNWs)により捕集されたCTCsの形態学的な解析を行うために、初期の乳がん患者から捕集したCTCsを走査電子顕微鏡で確認したし、5kV加速電圧のSEM(JSM−6701F、JEOL)により形態(morphology)を観察した。上記の捕集された細胞を2時間3.7%パラホルムアルデヒド(PFA)で固定して、脱水のために15分ごとにエタノールの濃度(50%、70%、90%、100%)を増加させながら露出した後、空気で完全に乾燥させた。サンプルをSEM顕微鏡で検査する前にplatinum goldでスパッタコーティングした。
抗体混合物が結合されたポリピロール磁性ナノワイヤ(Ab mixture_mPpyNWs)に捕集された細胞を回収するために、抗体混合物が結合されたポリピロール磁性ナノワイヤ(Ab mixture_mPpyNWs)に捕集された細胞に50mMのグルタチオン(GSH)溶液で60分間500rpmで振盪処理した。細胞の成長と増殖をモニタリングするために放出された細胞を24−well plateで接種した。
上記の実施例3の初期のがん患者のサンプルから捕集された血中のがん細胞(CTCs)のイメージ解析のために代表的な上皮マーカーを用いて固定した後、免疫染色を行ったが、この方法は複雑であり、時間が多く所要されて、多くの手続きを経なければならないので、現場肉眼検出戦略を導入して、迅速かつ信頼の高いがん細胞の存在を予測することができる簡単な比色免疫反応を図5aに図式して提示した。
抗体混合物が結合されたポリピロール磁性ナノワイヤ(Ab mixture_mPpyNWs)を用いて、がん患者の血液で捕集された血中のがん細胞(CTCs)のEGFR Exon 21 L858R遺伝子変異を解析するために、DNAを抽出した後digital PCRを用いて、がん組織で検出されたEGFR Exon 21 L858R遺伝子変異と比較した。
Claims (19)
- 導電性高分子を含む血中のがん細胞の検出および回収用のナノ構造体として、上記の導電性高分子は、抗体が結合されて磁性ナノ粒子が搭載された導電性高分子であることを特徴とする、血中のがん細胞の検出および回収用のナノ構造体。
- 上記の抗体は、anti−EpCAM(anti−Epithelial cell adhesion molecule)、anti−EGFR(anti−Epidermal growth factor receptor)、anti−N−cadherin、anti−TROP2(anti−trophoblast cell−surface antigen)およびanti−vimentinからなる群から選ばれるいずれか一つ以上であることを特徴とする、請求項1に記載の血中のがん細胞の検出および回収用のナノ構造体。
- 上記の抗体は、anti−EpCAM(anti−Epithelial cell adhesion molecule)、anti−EGFR(anti−Epidermal growth factor receptor)、anti−N−cadherin、anti−TROP2(anti−trophoblast cell−surface antigen)およびanti−vimentinを含む抗体混合物であることを特徴とする、請求項1に記載の血中のがん細胞の検出および回収用のナノ構造体。
- 上記の抗体混合物は、ホースラディッシュペルオキシダーゼ(horseradish peroxidase;HRP)をさらに含むことを特徴とする、請求項3に記載の血中のがん細胞の検出および回収用のナノ構造体。
- 上記の導電性高分子は、ポリアセチレン(polyacetylene)、ポリピロール(polypyrrole)、ポリチオフェン(polythiophene)、PEDOT(poly(3,4−ethylenedioxythiophene))、 ポリアニリン(polyaniline)またはこれらの誘導体であることを特徴とする、請求項1に記載の血中のがん細胞の検出および回収用のナノ構造体。
- 上記のナノ構造体は、ナノワイヤ、ナノロッド、またはナノ粒子であることを特徴とする、請求項1に記載の血中のがん細胞の検出および回収用のナノ構造体。
- 上記の血中のがん細胞は、血中循環腫瘍細胞(Circulating tumor cell;CTC)または血中循環がん幹細胞(Circulating Tumor Stem Cell;CTSC)であることを特徴とする、請求項1に記載の血中のがん細胞の検出および回収用のナノ構造体。
- 上記の血中のがん細胞は血中循環腫瘍細胞(Circulating tumor cell;CTC)であることを特徴とする、請求項7に記載の血中のがん細胞の検出および回収用のナノ構造体。
- (1)請求項1のナノ構造体を対象試料に処理するステップ;および
(2)磁石により生成される磁場を用いて、上記のナノ構造体から上記の血中のがん細胞を検出するステップを含む、血中のがん細胞の検出および回収方法。 - 化合物を用いて上記のナノ構造体から上記の血中のがん細胞を分離するステップをさらに含む、請求項9に記載の血中のがん細胞の検出および回収方法。
- 上記の化合物はグルタチオン(glutathione)であることを特徴とする、請求項10に記載の血中のがん細胞の検出および回収方法。
- 上記の試料は血液であることを特徴とする、請求項9に記載の血中のがん細胞の検出および回収方法。
- (1)ホースラディッシュペルオキシダーゼ(horseradish peroxidase;HRP)をさらに含む請求項1に記載のナノ構造体を対象試料に処理するステップ;および
(2)上記のナノ構造体の色を肉眼で判断するステップを含む、血中のがん細胞の比色検出方法。 - 上記のナノ構造体の色変化を分光計または色度計で測定して、対象試料内の血中のがん細胞の濃度を定量するステップをさらに含む、請求項13に記載の血中のがん細胞の比色検出方法。
- 上記の試料は血液であることを特徴とする、請求項13に記載の血中のがん細胞の比色検出方法。
- 請求項1に記載の血中のがん細胞の検出および回収用のナノ構造体を含む、がん診断キット。
- 上記の診断キットはバイオセンサーであることを特徴とする、請求項16に記載のがん診断キット。
- 請求項1に記載の血中のがん細胞の回収用のナノ構造体から検出した血中のがん細胞からDNAを抽出または分離して解析するステップを含む、がんの発病または予後を診断するための情報提供方法。
- 上記の解析は、試料の中のDNAの濃度、コピーの数または塩基配列を解析して遺伝子変異の可否を確認することを特徴とする、請求項18に記載の方法。
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CN111100839B (zh) * | 2018-10-29 | 2024-03-08 | 猎源(上海)生物医药科技有限公司 | EGFR/Vimentin/叶酸免疫脂质体磁球、制备方法及试剂盒 |
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KR102346111B1 (ko) * | 2020-02-07 | 2022-01-03 | 주식회사 싸이토딕스 | Ctc 분리용 마그네틱 비드 및 이를 이용한 ctc 분리방법 |
CN111856022A (zh) * | 2020-07-01 | 2020-10-30 | 山东凯歌智能机器有限公司 | 一种检测胰腺癌患者外周血循环肿瘤细胞E-Cadherin表达的试剂盒及检测方法 |
CN112011434B (zh) * | 2020-08-26 | 2022-01-04 | 武汉大学 | 一种用于富集循环肿瘤细胞的红细胞仿生涂层 |
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