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2 日中医学協会助成事業 シリカコーティング蛍光ナノ粒子を用いたセンチネルリンパ節生検並びにリンパ節到達様式に関する研究 研究者氏名 : 日本研究機関 : 指導責任者 : 叢莉蔓東北大学大学院医学系研究科 腫瘍外科学分野大内憲明教授 Abstract: Sentinel lymph node biopsy (SLNB) has been developed as a new diagnostic and therapeutic modality in melanoma and breast cancer surgery. There are two major methods for detection of SN using blue dye and radioisotope. However, there are disadvantages for each method. In this study we used nano-sized fluorescent beads of extremely narrow diameter distribution. This paper describes silica-coating of fluorescent microspheres by means of a seeded polymerization technique based on the Stöber method with modifying TEOS concentration at ~ 0.2M. We have successfully performed silica coating of fluorescent microspheres. We then demonstrated an application of silica-coated fluorescent beads for sentinel lymph node mapping by an animal model. Taken together, the chemical, optical and in vivo data presented in this study demonstrate the potential role of silica-coated fluorescent beads for imaging in medical treatments. Key words: sentinel lymph node biopsy, radioisotope method,silica-coated fluorescent nano-particle, Stöber method 背景 目的 : センチネルリンパ節生検はオーダーメイド医療を具現化する技術といえる 現在ラジオアイソトープ法 色素法の 2 種類が一般に施行されているが 前者は放射線管理区域でしか使用できないことから施設が限定されることや 少量とはいえ被爆の問題がある 後者は色素注入から切開まで適切な時間を要求され 客観的評価が困難である事 検出感度がやや低い事 体外診断できない事等それぞれ短所がある それらの欠点を補いうる新しいトレーサーが求められている 蛍光計測法はこれらの欠点を解決し得る新しいセンチネルリンパ節検出技術として期待され 蛍光ナノ粒子を新しいトレーサーとして利用する試みがなされている これまで我々は 粒径 100nm のポリスチレン蛍光ビーズのシリカコーティングに成功し 蛍光寿命の延長など蛍光特性の改善を認めた 本研究ではシリカコーティング法をナノ粒子の安定化技術として用いるべく 20nm および 40nm の蛍光ビーズのシリカコーティングを試み シリカによるカプセル化合成条件の探索を行い 更に動物実験を通じてセンチネルリンパ節の蛍光造影効果を検証した -86-
3 方法 : 蛍光波長 680nm で粒径 20nm および 40nm のポリスチレン蛍光ビーズを Stöber 法によりシリカコーティングした シリカ供給源である Tetraethylortsilicate(TEOS) の濃度を M の範囲で変え, 最適な条件を検討した 動物はラット ( ドンリュウ, オス 5-7 週齢 体重 g) をそれぞれの粒子につき 5 匹 10 肢ずつ実験に用いた シリカコーティング蛍光ナノ粒子をラット後肢足背に皮下注射後 鼠径部および膝窩部にレーザー光を当て 分後にそれぞれを蛍光計測した その後リンパ節を摘出し 蛍光の確認 透過電子顕微鏡 (TEM) による蛍光ナノ粒子並びに組織観察 従来のパテントブルーを用いた色素法との比較検討を行った さらに シリカコーティング蛍光ナノ粒子及び色素法の間で検出感度を比較した 結果 : TEOS 0.02M で厚さ 10nm のシリカ層を生成し 20nm および 40nm のビーズからそれぞれ粒径 40nm および 60nm のナノ粒子を作製できることがわかった センチネルリンパ節造影例においては 粒径 40nm および 60nm のシリカコーティング蛍光ナノ粒子のラット足背皮下投与後双方 1 分後には下肢の皮下リンパ管が造影され 30 分後には鼠径または膝窩のどちらかのリンパ節が造影されはじめ 60 分後には鼠径および膝窩リンパ節が明確に確認できた 120 分後もリンパ管及びリンパ節全体の造影が維持され コーティングしない粒子に比べて造影時間の延長を認めた 粒径 40nm のシリカコーティング蛍光ビーズのリンパ節検出感度は 50% であり色素法とほぼ同等であり 粒径 60nm のシリカコーティング蛍光ナノ粒子の検出率 70% と高いことがわかった TEM による観察では 40nm 及び 60nm のシリカコーティング蛍光ナノ粒子は双方に観察されたことからマクロファージに貪食されて受動的にリンパ節に運ばれるものと直接リンパ管をリンパ流に乗って輸送されるものの両方が存在することがわかった 左図 : シリカコーティング蛍光ナノ粒子を用いたラットのセンチネルリンパ節の蛍光計測検出画像 右図 : 60nm シリカコーティング蛍光ナノ粒子がマクロファージに取り込まれていた -87-
4 考察 : 粒径 40nm および 60nm のシリカコーティング蛍光ナノ粒子は遊走性貪食細胞であるマクロファージ並びにリンパ節内在性貪食細胞である樹状細胞の双方に観察されたことから直接リンパ管を通ってリンパ節の樹状細胞に取り込まれて造影効果が得られる場合といったんマクロファージに取り込まれからマクロファージの移動によるリンパ節の造影効果が得られる場合の二通りの輸送様式が存在することが強く示唆された シリカコーティング技術は蛍光ナノ粒子を保護することで蛍光寿命の改善 安全性確保 またサイズ調節による組織滞留性の向上等の点から様々な応用が考えられる 本研究により TEOS 濃度を変えることで微細なナノ粒子のコーティングを行い得ることがわかった 今後の展開として 大型動物モデルによる有用性並びに長期を含めた安全性の確認 更に シリカ層表面への機能分子修飾による応用の拡大を目指している シリカコーティング蛍光ナノ粒子は簡便で低侵襲な画像診断を実現する上で極めて有用な技術を考えられる 参考文献 : 1. Haagensen CD. Diseases of the Breast, 2nd ed. Philadelphia, W.B. Saunders, , Cabanas R. An approach for the treatment of penile carcinoma. Cancer. 39: , Borgstein P, Meijer S. Historical perspective of lymphatic tumour spread and the emergence of the sentinel node concept. Eur J Surg Oncol. 24: 85-95, Noguchi M, Tsugawa K, Bando E. Sentinel lymphadenectomy in breast cancer; Identification of sentinel lymph node and detection of metastases. Breast Cancer Res Treat. 53: , 三輪佳宏. 蛍光 発光試薬の選び方と使い方. 羊土社 , 草野満夫.LED 励起 ICG 蛍光をトレーサーとした新しいセンチネルリンパ節同定法 消化器癌への応 用. 日消外会誌, 39:1464, Kataoka K. Block copolymer micelles for drug delivery: design characterization and biological significance Adv Drug Deliv Rev. 47: ,
5 8. Kobayashi Y, Misawa K, Kobayashi M, Takeda M, Konno M, Satake M, Kawazoe Y, Ohuchi N, Kasuya A. Silica-coating of fluorescent polystyrene microspheres by a modified Stöber method and their stability against photo-bleaching. e-polymers. 52: 1-8, Nakajima M, Takeda M, Kobayashi M, Suzuki S, and Ohuchi N. Nano - sized fluorescent particles as new tracers for sentinel node detection: experimental model for decision of appropriate size and wavelength. Cancer Sci. 96: , Kobayashi Y, Misawa K, Takeda M, Ohuchi N, Kasuya A, Konno M. Control of Shell Thickness in Silica-Coating of AgI Nanoparticles. Adv Mater Res : , 注 : 本研究は 2008 年 5 月 7 日 ~9 日 ナノ学会第 6 回大会 にて口頭発表 PCEM Tohoku Univ. Med. School (V0l. 28, ) に掲載 作成日 :2009 年 3 月 13 日 -89-
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!!!!!!!!!!!! TEM! SEM, AFM! 参考文献 最先端材料システム One Point! イメージング (共立出版) 実験がうまくいく 蛍光 発光試薬の 選び方と使い方! (羊土社) 非侵襲 可視化 技術ハンドブック (NTS) 講義と実習 生細胞蛍光 イメージング (共立出版) よくわかる生物電子 顕微鏡技術 (共立出版) (in vivo) - /! - MRI! - X! in
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