JP2013505728A - 細胞を磁性化するための材料及び磁気操作 - Google Patents
細胞を磁性化するための材料及び磁気操作 Download PDFInfo
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Abstract
Description
本出願は、2009年9月25日に出願の米国仮特許出願第61/245846号の優先権を主張し、またその全体は参照することにより本明細書に組み入れられる。
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図1は、負のナノ粒子(1)及び正のナノ粒子(3)(少なくとも1つのナノ粒子は磁気を帯びている)及び支持分子(5)を組み合わせることにより、磁性ナノ粒子アセンブリ(7)を調製する一般的な模式図を示すものである。
本明細書において我々は、Fe2O3ナノ粒子のみ(<50nm粒子サイズ)、Au−(Fe2O3)ナノ粒子、及びPL−AU−(Fe2O3)の完全な磁性ナノ粒子アセンブリについて試験することにより、磁性ナノ粒子アセンブリの様々な構成要素の重要性についてもまた実証した。
我々はまた、磁性ナノ粒子アセンブリが材料を保持する、及び材料を細胞に送達する能力について示した。原理の証明として、HEK293細胞をGFP DNAを有する磁性ナノアセンブリを用いて処理及び浮遊させ、その後、浮遊した3D細胞培養中の細胞の内部でのGFPの蛍光シグナルを検出した。
多くの場合、in vitroで初代細胞を培養することは難しいものであるが、我々は酸化鉄−Au−PLを用いて初代細胞を磁性化した後に、磁気浮遊により3Dにおいて、それらの培養を成功させた。
図7を参照すると、血液、血清、血漿、又は凝集していない組織細胞(43)を含むがこれらには限定されない、動物又はヒトから採取した任意の細胞(41)をナノ粒子(45)と混合し、その後、それらを共に30秒から48時間インキュベートする。インキュベートの間に、この試料(43)及びナノ粒子(43)は、ナノ粒子アセンブリ(47)を形成するように、試料及びナノ粒子中に含まれる任意のタンパク質、DNA又はポリサッカライドと静電的に相互作用する。次にこの磁性ナノ粒子アセンブリ(47)を磁力、遠心分離、及び/又は沈降により分離させる。この過程で上清を混合物から分離し、そして残った磁性化細胞を操作するために磁石を用いる。
本明細書において我々は、磁性化した際に細胞を凍結し、その後融解して3D培養に用いることができることを示した。上述の方法により、細胞を磁性ナノ粒子アセンブリと混合する。過剰の磁性ナノ粒子アセンブリをその後除去し、標準的な方法に従い、細胞を洗浄及び凍結保存する。その後細胞を融解し、3D培養系において培養する。この方法により、研究及び治療で使用するための磁性化細胞の調製及び商品化が可能になるため、これは非常に便利である。
我々は又、細胞又は細胞抽出物を既にある3D培養に加えることにより、複数回の細胞浮遊を行うこともできる。このようにして、3D培養が成長するにつれ、培養中に様々な細胞又は細胞産物を濃縮することができる。
上述の実施形態において我々は、この構成要素の緊密な混合物を提供するように、磁性ナノ粒子アセンブリと細胞を混合した。しかしながら、我々はまた、緊密な混合は必要ではなく、単に磁性ナノ粒子アセンブリに隣接するだけで、細胞は磁性ナノ粒子を取り込むであろうことを示した。このことは、細胞が磁性ナノ粒子アセンブリ材料を含まないことが必要な場合に有益である。
(1)Shimizu,K.;Ito,A.;Arinobe,M.;Murase,Y.;Iwata,Y.;Narita,Y.;Kagami,H.;Ueda,M.;Honda,H. J.Biosci.Bioeng.2007,103,472−8.
(2)Kosmulski,M.;Marcel Dekker:Chemical Properties of Material Surfaces:New York, 2001.
(3)Mahmoudi,M.;Simchi,A.;Imani,M. J. Iran. Chem. Soc. 2010, 7, Sl−S27.
(4)Bacri,J.−C.;Perzynski,R.;Salin,D.;Cabuil,V.;Massart,R. J. Magn. Magn. Mater. 1990, 85, 27−32.
(5)Douziech−Eyrolles,L.;Marchais,H.;Herve,K.;Munnier,E.;Souce,M.;Linassier,C.;Dubois,P.;Chourpa,I. Int. J. Nanomed. 2007, 2, 541−550.
(6)Duff,D.G.;Baiker,A.;Edwards,P.P. Langmuir 1993, 9, 2301−2309.
米国特許第2005054101号、国際公開第2005010162号
米国特許第2009137018号、国際公開第2005003332号
米国特許第2006063252号、国際公開第2004083412号、国際公開第2004083416号
国際公開第2010036957号
Claims (20)
- 細胞を磁性化するための組成物であって、
a)負に帯電したナノ粒子;
b)正に帯電したナノ粒子;及び
c)支持分子、
を含み、ここで前記負に帯電したナノ粒子又は正に帯電したナノ粒子のうちの1つが磁気応答性エレメントを含み、かつ、緊密な混合状態で前記支持分子が前記負に帯電したナノ粒子及び前記正に帯電したナノ粒子を保持する組成物。 - 前記負に帯電したナノ粒子が金のナノ粒子である、請求項1に記載の組成物。
- 前記正に帯電したナノ粒子が酸化鉄のナノ粒子である、請求項1に記載の組成物。
- 前記支持分子がペプチド、ポリサッカリド、核酸、ポリマー又はその組み合わせを含む、請求項1に記載の組成物。
- 前記支持分子が、ポリ−リシン、フィブロネクチン、コラーゲン、ラミニン、BSA、ヒアルロナン、グリコサミノグリカン、アニオン性の非硫酸化グリコサミノグリカン、ゼラチン、核酸、細胞外マトリックスタンパク質、細胞抽出物、抗体又はその混合物若しくは誘導体を含む、請求項1に記載の組成物。
- 前記支持分子がポリ−リシンを含む、請求項1に記載の組成物。
- 前記細胞が動物の組織又は体液から得られたものであり、かつ、前記支持分子が前記組織又は体液により提供される、請求項1に記載の方法。
- さらに細胞を含む、請求項1に記載の組成物。
- a)前記支持分子がペプチド、ポリサッカライド、核酸、ポリマー、ポリ−リシン、フィブロネクチン、コラーゲン、ラミニン、BSA、ヒアルロナン、グリコサミノグリカン、アニオン性非硫酸化グリコサミノグリカン、ゼラチン、核酸、細胞外マトリックスタンパク質混合物、抗体、又はその混合物若しくは誘導体を含み、
b)前記負に帯電したナノ粒子が金のナノ粒子であり、及び
c)前記正に帯電したナノ粒子が酸化鉄のナノ粒子である、請求項1に記載の組成物。 - さらに細胞を含む、請求項9に記載の組成物。
- 請求項1に記載の組成物と細胞を接触させること、及び該細胞が磁性化するまで1〜12時間インキュベートすること、及び磁性化した前記細胞を、前記細胞を移動させるのに十分な磁場にかけることを含む、細胞を移動させる方法。
- 前記磁場が非対称である、請求項11に記載の方法。
- 前記細胞が懸濁されている又は接着している、請求項11に記載の方法。
- 細胞懸濁液と請求項8に記載の組成物とを混合することを含む、請求項11に記載の方法。
- 前記磁性化細胞を、前記細胞を移動させるのに十分な磁場にかける前に、前記組成物を除去するために前記細胞を洗浄することをさらに含む、請求項11に記載の方法。
- 標的分子を加えた請求項1に記載の組成物と細胞懸濁液とを接触させること、前記細胞が磁性化し、前記標的が前記細胞に送達されるまで1〜12時間インキュベートすること、及び磁性化した前記細胞を3D培養するために、磁場を磁性化した前記細胞にかけることを含む、3D培養用に標的分子を細胞に送達する方法。
- 請求項1に記載の組成物とインキュベートすることにより作成される磁性化細胞を含む組成物。
- 請求項9に記載の組成物とインキュベートすることにより作成される磁性化細胞を含む、請求項17に記載の組成物。
- 前記細胞が凍結している、請求項17に記載の組成物。
- 前記細胞が凍結している、請求項18に記載の組成物。
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