JP6925984B2 - 無核細胞への物質の送達 - Google Patents
無核細胞への物質の送達 Download PDFInfo
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- JP6925984B2 JP6925984B2 JP2017565959A JP2017565959A JP6925984B2 JP 6925984 B2 JP6925984 B2 JP 6925984B2 JP 2017565959 A JP2017565959 A JP 2017565959A JP 2017565959 A JP2017565959 A JP 2017565959A JP 6925984 B2 JP6925984 B2 JP 6925984B2
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Description
本出願は、2015年7月9日に出願された米国仮特許番号第62/190,677号の優先権を主張し、その全体は参照により本明細書に援用される。
本発明は少なくとも一部が、National Institute of Health(NIH)によって授与されたR01GM101420−01A1及びNIHによって授与されたRC1EB011187−02の下で政府助成により行われた。米国政府は本発明において一定の権利を有する。
本明細書において使用される時、「マイクロ流体システム」は、低体積(例えばμL、nL、pL、fL)の流体が、小体積の液体の離散的な処理を達成するようにプロセシングされるシステムを指す。本明細書において記載されるある特定の実装には、マルチプレックス化、自動化、及びハイスループットスクリーニングが含まれる。流体(例えばバッファー、溶液、ペイロード含有溶液、または細胞懸濁物)は、移動されるか、混合されるか、分離されるか、またはそうでなければプロセシングされ得る。本明細書において記載されるある特定の実施形態において、マイクロ流体システムを使用して、バッファー中に懸濁された細胞へ機械的狭窄を適用し、ペイロードまたは化合物が細胞のサイトゾルに入ることを可能にする細胞における摂動(例えば穴)を誘導する。
本明細書において使用される時、「無核細胞」は、核を欠損する細胞を指す。かかる細胞には、血小板、赤血球(red blood cell)または赤血球(erythrocyte)(本明細書において互換的に使用される)、及び網状赤血球が含まれ得るが、これらに限定されない。網状赤血球は、典型的にはヒト身体中の赤血球の約1%を構成する未成熟(例えば、まだ両凹面でない)赤血球である。網状赤血球も無核である。ある特定の実施形態において、本明細書において記載されるシステム及び方法は、無核細胞(例えばRBC、網状赤血球、及び/または血小板)の、濃縮された(例えば天然で見出されるよりも高いパーセンテージの全細胞集団を含む)集団、精製された集団、または単離された(例えばそれらの天然環境から、実質的に純粋なまたは均一の形状で)集団の処理及び/またはプロセシングに使用される。ある特定の実施形態において、本明細書において記載されるシステム及び方法は、RBC、網状赤血球、血小板に加えて、他の血球を含有する全血の処理及び/またはプロセシングに使用される。これらの細胞タイプの精製または濃縮は、公知の方法(密度勾配システム(例えばFicoll−Hypaque)、蛍光活性化細胞選別(FACS)、磁気細胞選別、または赤芽細胞及び赤芽球前駆細胞のインビトロの分化等)を使用して達成される。
上述のように、RBCの形は他の哺乳類細胞とは異なる。それらの非対称性の両凹面の形の結果として、RBCは異なる方向で狭窄に入り通過する能力を有する。この特性は、他の細胞(懸濁中にある場合多かれ少なかれ球形を有する)で存在しない。これは送達の観点から予想外の要素である。例えば、RBCの最も幅広い寸法で、本明細書において記載されるマイクロ流体デバイスの狭窄を介して進むRBCは、細胞膜が破壊される可能性がより高く、一方で、RBCの最も狭い寸法で、狭窄を介して進むRBCは細胞膜が破壊される可能性はより低い。したがって、本明細書において記載されるマイクロ流体デバイス及びシステムの狭窄の寸法の範囲は、かかる特有な特徴及び寸法(例えばRBC等の無核の非対称の細胞)の細胞により作業する際の困難さを克服するために、初期の立体配置よりも少ない。いくつかの実施形態において、無核細胞は、マイクロ流体チャネルが少なくとも約6μm以下の幅を備えた狭窄を含む、本明細書において記載されるマイクロ流体システムによりプロセシングされる。例えば、RBCは、約1、2、3、4、5、または約6μmの狭窄の幅によりプロセシングされ得る。他の実施形態において、狭窄の幅は1μm〜4μmである。更なる実施形態において、狭窄の幅は2μm〜4μmである。
無核細胞へ様々な化合物を送達することができるマイクロ流体システムが構築された。後述されるように、これらのマイクロ流体システムは、ペイロード(例えば化合物またはマクロ分子)が細胞膜を介し、サイトゾルの中へ通過し、細胞送達をもたらすのに十分な大きさの細胞の摂動を引き起こすように構成された狭窄を介して、バッファー中に懸濁された無核細胞が通過することを可能にするように構築された。図1A及び図1B中で示されるように、マイクロ流体システム(5)は管状内腔を画成するチャネルを含む。マイクロ流体チャネルは、単一の細胞(20)のみが一度で狭窄を介して通過することができるように構成できる狭窄(15)を含む。細胞は、送達物質(30)も含むバッファー(25)中に懸濁されているチャネルを介して通過することができるが、細胞が狭窄を介して通過する前または後に、送達物質をバッファーへ添加することができる。細胞が狭窄に接近し通過する時、狭窄は細胞へ圧力(例えば機械的圧縮)を適用し、それによって細胞(細胞201として示される)を圧迫する。狭窄によって細胞へ適用された圧力は、細胞膜中に摂動(例えば穴)を引き起こし、細胞(202)の摂動をもたらす。一旦細胞が狭窄を介して通過すると、摂動された細胞は、摂動を介してバッファー中の物質(送達物質が含まれる)を取り込み始め、細胞(203)の送達をもたらす。細胞膜は経時的に回復し、少なくとも送達物質の一部分は細胞の内部で捕捉されたままである。
物質をヒトRBCへ送達する本明細書において記載されるマイクロ流体デバイスの能力を、蛍光標識されたデキストランポリマー及び蛍光融合タンパク質を使用して試験した。簡潔には、ヒトRBCを新鮮血から単離し、完全増殖培地中で培養した。RBCをP.falciparumにより感染させた。細胞を4つの異なる感染後の時点で採取した。図3Cは、ギムザにより染色された赤血球サンプルの薄フィルム塗抹標本を示し、様々な増殖期のマラリア原虫を強調する。時点1はリング期寄生生物(侵入後<18時間)を反映し、時点2はトロホゾイト期寄生生物(<T1+8時間)を反映し、時点3はトロホゾイト期寄生生物(T1+22時間)、時点4はシゾント期寄生生物(T1+30時間)を反映する。感染の異なる期のRBCを示された時点で採取し、1x108細胞/mLの密度でPBS中に懸濁した。P.falciparumへ曝露された所定のサンプルは、感染細胞及び非感染細胞の両方を有していた。70kDaのフルオレセインに標識されたデキストランまたは10kDaのAPCに標識されたデキストランを、2つのデザインのうちの1つによるマイクロ流体システムを使用して、感染RBC及び非感染RBCの懸濁液へ送達した。1つのマイクロ流体システムデザインは、10μmの長さ及び4μmの幅を備えた単一の狭窄を含んでいた(10×4)。他のマイクロ流体システムデザインは、各々30μmの長さ及び5μmの幅を備えた、並列して配置された5つの狭窄を含んでいた(30×5×5)。
Claims (13)
- 無核細胞の細胞膜における摂動を引き起こすためのマイクロ流体システムであって、
内腔を画成し、バッファー中に懸濁された無核細胞がそれを介して通過できるように構成される、少なくとも1つのマイクロ流体チャネルを含み、前記マイクロ流体チャネルが、長さ、深さ、及び幅を含む少なくとも1つの細胞変形狭窄を含み、前記狭窄の幅が4μm未満である、前記マイクロ流体システムを用いて、無核細胞内にペイロードを送達するための方法であって、以下のステップを含む、前記方法。
圧力を無核細胞へ適用してペイロードの通過に十分な大きさの前記無核細胞の摂動を引き起こすように、バッファー中に懸濁された前記無核細胞を、前記細胞変形狭窄を含む前記マイクロ流体チャネルを介して通過させるステップ;及び
前記無核細胞が前記狭窄を介して通過する前または後に所定時間にわたってペイロード含有溶液中で前記無核細胞をインキュベーションするステップ; - 無核細胞が
(i)健康な無核細胞である;または
(ii)疾患無核細胞若しくは感染無核細胞である;
請求項1に記載の方法。 - 無核細胞が、赤血球、網状赤血球、または血小板のうちの1つまたは複数である、請求項1または2に記載の方法。
- (i)幅が0.5μm〜4μmである;
(ii)長さが30μm以下である;
(iii)深さが1μm〜1mmである;
(iv)バッファーが無核細胞を膨潤させる低張バッファーである;及び/または
(v)チャネルの横断面が、円形、楕円形、細長いスリット、正方形、六角形、及び三角形からなる群から選択される;
請求項1〜3のいずれか一項に記載の方法。 - (i)幅が1μm〜3μmである;
(ii)長さが10μm〜30μmである;及び/または
(iii)深さが1μm〜20μmである;
請求項4に記載の方法。 - (i)幅が無核細胞の最大直径未満である;
(ii)長さが10μm〜20μmである;及び/または
(iii)深さが20μmである;
請求項4または5に記載の方法。 - 幅が無核細胞の最大直径の20%〜99%である、請求項4〜6のいずれか一項に記載の方法。
- マイクロ流体システムが、複数のマイクロ流体チャネル及び/または複数の細胞変形狭窄を含む、請求項1〜7のいずれか一項に記載の方法。
- (i)マイクロ流体システムが、並列または連続する複数のマイクロ流体チャネルを含む;
(ii)マイクロ流体システムが、複数の細胞変形狭窄を含み、
前記複数の細胞変形狭窄が同じマイクロ流体チャネル中で並列して配置される;及び/または
(iii)マイクロ流体システムが、複数の細胞変形狭窄を含み、
前記複数の細胞変形狭窄が同じマイクロ流体チャネル中で連続して配置される;
請求項8に記載の方法。 - マイクロ流体システムが、細胞駆動装置をさらに含み、
(i)前記細胞駆動装置が、バッファーへ90psiを超える圧力を適用するように適合される;及び/または
(ii)前記細胞駆動装置が、圧力ポンプ、ガスボンベ、コンプレッサー、真空ポンプ、シリンジポンプ、蠕動ポンプ、ピペット、ピストン、キャピラリーアクター、ヒト心臓、ヒト筋肉、重力、マイクロ流体ポンプ、及びシリンジからなる群から選択される;
請求項9に記載の方法。 - ペイロード含有溶液が、タンパク質、ペプチド、小分子、核酸、脂質、炭水化物、マクロ分子、ビタミン、ポリマー、蛍光色素、フルオロフォア、カーボンナノチューブ、量子ドット、ナノ粒子、及び/またはステロイドのうちの1つまたは複数を含む、請求項1〜10のいずれか一項に記載の方法。
- ペイロード含有溶液が、
(i)タンパク質またはポリマーを含む;
(ii)タンパク質及びポリマーを含む;
(iii)小分子またはタンパク質を含む;
(iv)小分子及びタンパク質を含む;あるいは
(v)クロロキン、アトバクオン−プログアニル、アルテメテル/リメファントリン(lymefantrine)、キニーネ硫酸塩、メフロキン、ヒドロキシクロロキン、プリマキン、キニジン、アルテスネート、アルテミシニン、スルファドキシン/プリメサミン(pryimethamine)、アモジアキン、スルホンアミド、ハロファントリン、ドキシサイクリン、テトラサイクリン、クリンダマイシン、ヒドロキシ尿素、ハイドレア、ビタミンE、L−グルタミン、アシクロビル、ガンシクロビル、バラシクロビル、ペンシクロビル、トリ−ペプチド、またはテトラ−ペプチドのうちの1つまたは複数を含む;
請求項11に記載の方法。 - ペイロードが細胞内に含有されるように、無核細胞の摂動が停止するのを所定時間にわたって待機するステップをさらに含む、請求項1〜12のいずれか一項に記載の方法。
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CN107922911A (zh) | 2015-07-09 | 2018-04-17 | 麻省理工学院 | 将物质递送至无核细胞 |
EP3344747B1 (en) | 2015-09-04 | 2022-11-09 | SQZ Biotechnologies Company | Intracellular delivery of biomolecules mediated by a surface with pores |
EP3344575B1 (en) | 2015-09-04 | 2020-04-15 | SQZ Biotechnologies Company | Intracellular delivery of biomolecules to cells comprising a cell wall |
JP2018538343A (ja) | 2015-12-22 | 2018-12-27 | サングイ バイオ ピーティーワイ. エルティーディー | 赤血球を用いる治療方法 |
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CN109415741A (zh) | 2016-05-03 | 2019-03-01 | Sqz生物技术公司 | 细胞内递送生物分子以诱导耐受性 |
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JP7255904B2 (ja) | 2023-04-11 |
JP2021176334A (ja) | 2021-11-11 |
EP3320082B1 (en) | 2023-05-24 |
CA2988996A1 (en) | 2017-01-12 |
AU2021212058A1 (en) | 2021-08-26 |
AU2016289530A1 (en) | 2018-01-18 |
US20220195364A1 (en) | 2022-06-23 |
EP4257675A2 (en) | 2023-10-11 |
EP3320082A4 (en) | 2019-02-27 |
EP4257675A3 (en) | 2024-01-03 |
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