JP2021137005A - マクロファージの細胞付着・再生促進用ナノリガンド、及びこれを用いたマクロファージの細胞付着・再生を促進する方法 - Google Patents
マクロファージの細胞付着・再生促進用ナノリガンド、及びこれを用いたマクロファージの細胞付着・再生を促進する方法 Download PDFInfo
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- 231100000765 toxin Toxicity 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
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Abstract
Description
マクロスケールのナノリガンド密度の可逆的変化を引き起こせないという短所があった。
インテグリン付着性リガンドペプチドは、負に荷電されたことを特徴とする、マクロファージの細胞付着・再生促進用ナノリガンドを提供する。
コアをリンカーを含む第1の懸濁液と混合してリンカーが結合されたコアを製造するステップと、
リンカーが結合されたコアをインテグリン付着性リガンドペプチド(RGD)を含む第2の懸濁液と混合するステップと、を含む、上述のマクロファージの細胞付着・再生促進用ナノリガンドの製造方法を提供する。
製造例1
スライド可能なナノリガンド(slidable nano−ligand)の製造
1)磁性コアの製造(MNP)
スライド可能なナノリガンドの磁気制御のために、スライド可能なナノリガンドの磁性コアを下記のように製造した。エタノール約80mL、脱イオン水(DI)60 mL、ヘプタン140 mLをまず混合した。この混合物に、不活性環境下で120mmolのオレイン酸ナトリウム(sodium oleate)と40mmolの塩化鉄(III)六水和物(iron(III)chloride hexahydrate)をエタノール80mL、脱イオン水(DI)60mL及びヘプタン140mLの溶媒混合物に70℃で4時間溶解させた。オレ酸鉄を含むヘプタン層を回収し、脱イオン水で洗浄した。ヘプタンを蒸発させて乾燥させたオレ酸鉄を回収し、ここにオレイン酸20mmolと1−オクタデセン200gを乾燥オレ酸鉄約40mmolに添加した。この溶液を100℃で5分間攪拌した。続いて、温度を320℃まで上げ、約30分間維持した。上記の混合物溶液を大気下で懸濁させ、室温に冷却した後、永久磁石を用いてエタノールで3回洗浄し、ナノ粒子を回収した。この磁性コアナノ粒子(MNP)を保存するために、溶液を使用時までヘプタンに保存した。
磁性コアナノ粒子に、スライド可能なナノリガンドのナノアセンブリのため、アミノシリカシェルでコーティングを施した。磁性コアナノ粒子(30mg)をシクロヘキサン(25mL)に分散させた。上記の懸濁液にトリトン−X(Triton−X)(5mL)、1−ヘキサノール(5mL)、NH4OH(0.5mL)、脱イオン水(1mL)を順次添加して30分間攪拌した。エマルジョンを安定化させた後、テトラエチルオルトシリケート(TEOS、12.5μL)を徐々に混合し、10分間攪拌した。アミノ官能基化のために(3−アミノプロピル)トリエトキシシラン(APTES、6.25mL)を混合し、一晩撹拌した。アミノ官能基化の後、アミノシリカシェルでコーティングされたMNPをそれぞれ3回ずつ、アセトン及びジメチルホルムアミド(DMF)で洗浄し、磁石を用いて回収した。
ポリエチレングリコール(PEG)リンカーを用いて、ナノリガンドのスライド特性を向上させ、荷電されたRGDペプチドリガンドを表面がアミノシリカシェルでコーティングされたMNPにグラフトした。PEGリンカーは、スライド可能なナノリガンドのナノアセンブリからの細胞吸収を防止するために使用された。DMF(1mL)中、アミノシリカでコーティングされたMNPを使用し、5mgのマレイミド− ポリ(エチレングリコール)−NHSエステル(Mal−PEG−NHS ester;Mn=5000Da、Biochempeg)を溶解させた。N、N−ジイソプロピルエチルアミン(DIPEA、2μL)を添加し、一晩撹拌した後、DMFで3回、ジメチルスルホキシド(DMSO)で3回洗浄し、永久磁石を使用して回収した。DMSO(1mL)中、PEG−アミノシリカでコーティングされたMNPを添加し、負に荷電されたチオール化RGDペプチド(CDDRGD、GL Biochem、0.5 mg)を溶解させた。二硫化物結合の形成を防止するために、DIPEA(2μl)とトリス(2−カルボキシエチル)ホスフィン塩酸塩(Tris(2−carboxyethyl)phosphine hydrochloride、TCEP、10mM)をこの懸濁液に添加し、一晩、暗条件の下で攪拌した。懸濁液をDMSOで洗浄し、静電相互作用を介して結合する基板が結合される前に、永久磁石を使用して回収した。
負に帯電されたチオール化RGDペプチド(CDDRGD、GL Biochem)を添加していないことを除いて、製造例1と同様の方法で「No RGD」ナノリガンドを製造した。
実施例1
スライド可能なナノリガンド、並びに当該ナノリガンドと基板との組み合わせ
上記の製造例で製造したスライド可能なナノリガンドを基板に可逆的に結合させるために、培養グレードガラスカバースリップ(culture−grade glass coverslips、22 mm x 22 mm)を使用した。基板を30分間塩酸とメタノールの1:1混合物に浸漬させ、表面の有機汚染物を除去されたことを確認した後、脱イオン水で3回洗浄した。これを硫酸で1時間浸漬させ、脱イオン水で洗浄した。基板を暗条件の下で1時間APTESとエタノールの1:1混合物に浸漬させ、基板をアミノ化させた。次いで基板をエタノールで3回洗浄し、100℃で1時間乾燥させた。静電相互作用を促進するために、1mLのDMSOにおいて、負に荷電されたスライド可能なナノリガンド(RGD−PEG−アミノシリカシェルでコーティングされた磁性ナノ粒子)をDMSO(1:20)で希釈し、超音波処理を施し、正に荷電されたアミノ化基板と1時間培養した。基板を脱イオン水で洗浄し、スライド可能なナノリガンドが存在する基板を得た。
実験例1
本発明によるスライド可能なナノリガンドの形態を確認するため、スライド可能なナノリガンドを対象に透過型電子顕微鏡(TEM)、動的光散乱・高角散乱環状暗視野走査透過型電子顕微鏡(HAADF−STEM)分析を行い、その結果図3及び図4に示した。
本発明によるスライド可能なナノリガンドのIn situ可逆的・時空間的制御を実証するために、スライド可能なナノリガンドを対象に走査型電子顕微鏡(SEM)で撮影し、原子間力顕微鏡(AFM)イメージングを行い、その結果を図8及び図9に示した。
目的とし、AFM撮像(Asylum Research、XE− 100 System)を25℃にて大気モードのACで行った。バネ定数(spring constant、5−3N/m)と共振周波数(resonance frequency、96〜175 kHz)を有するAFMカンチレバー(cantilever)(Nanosensors、SSS−SEIHR−20)を使用した。基板の近傍において磁石のない静的シリアル撮像を、スライド可能なナノリガンドの磁石介在変位を検査するため、基板の同スキャン領域において行った。In situ磁気撮像のために、まず撮像を行って、永久磁石を基板の底やスキャン領域の反対側に配置し、In situナノスケールのナノリガンドスライドを特徴づけられるよう、同スキャン領域における撮像を行った。その結果は、図8及び図9に示した。
本発明によるスライド可能なナノリガンドを用いたマクロファージの細胞付着や再生を促進する方法で、マクロスケールのナノリガンドの密度を調整することがマクロファージの細胞付着調整に及ぼす影響を確認するために、次のような実験を行った。
本発明によるナノリガンドのマクロスケールの密度調整がマクロファージの可逆的な付着を制御しているか否かに関する実験を下記のように行った。
本発明によるスライド可能なナノリガンドが、時間制御調整(tuning)により、マクロファージの細胞付着依存分極を調整できることを確認するために、次のように実験を行った。その結果を図16〜20に図示した。
本発明によるスライド可能なナノリガンドはIn situ制御により体内宿主マクロファージの細胞付着及び表現型を空間的に調整することを確認するために、下記のような実験を行い、その結果を図21〜図23に図示した。
深部の内部組織で組織の修復を促進し、炎症や繊維カプセルの形成を抑制することにおいて、磁場を介在した時空間的、可逆的、長期的調整に関する実験を行い、臨床環境でインプラントの免疫調整が可能であることを確認した。
Claims (12)
- 磁性ナノ粒子を含むコアと、前記コアを包み込むように設けられ、インテグリン付着性リガンドペプチドを含むコーティング層と、を含み
前記インテグリン付着性リガンドペプチドは、負に荷電されたことを特徴とする、マクロファージの細胞付着・再生促進用ナノリガンド。 - 前記磁性ナノ粒子は、シリカが表面に結合されたことを特徴とする、請求項1に記載のマクロファージの細胞付着・再生促進用ナノリガンド。
- 前記コアとコーティング層を接続するリンカーを更に含み、
前記リンカーは、ポリエチレングリコール(PEG)系リンカーである、請求項1に記載のマクロファージの細胞付着・再生促進用ナノリガンド。 - 前記ナノリガンドは、直径が30nm〜60nmであり、前記磁性ナノ粒子は、直径が5nm〜30nmである、請求項1に記載のマイクロファージの細胞付着・再生促進用ナノリガンド。
- 前記インテグリン付着性リガンドペプチドは、負に荷電されたチオール化インテグリン付着性リガンドペプチドを含み、
前記ナノリガンドの表面は負に荷電されている、請求項1に記載のマイクロファージの細胞付着・再生促進用ナノリガンド。 - 磁性ナノ粒子を含むコアを用意するステップと、
コアをリンカーを含む第1の懸濁液と混合してリンカーが結合されたコアを製造するステップと、
前記リンカーが結合されたコアをインテグリン付着性リガンドペプチド(RGD)を含む第2の懸濁液と混合するステップと、を含む、請求項1〜5のうちいずれか1項に記載のマクロファージの細胞付着・再生促進用ナノリガンドの製造方法。 - 請求項1〜5のうちいずれか1項に記載のマクロファージの細胞付着・再生促進用ナノリガンドを含む溶液に、表面が正に荷電された基板を担持して、ナノリガンド提示基板を製造するステップと、
前記ナノリガンド提示基板に培養液を処理した後、外部の磁場を印加し、マクロファージの細胞付着・再生性分極化を調整するステップと、を含む、マクロファージの細胞付着・再生を促進する方法。 - 前記ナノリガンド提示基板を製造するステップは、
基板の表面を酸性溶液に浸漬させるステップと、
浸漬済みの基板をアミノシラン溶液に担持して前記基板の表面が正に荷電させるステップと、
正に荷電された基板を室温で超音波を利用して処理するステップと、を含む、請求項7に記載のマクロファージの細胞付着・再生を促進する方法。 - 前記表面が正に荷電された基板は、アミノシラン溶液に基板を担持して前記基板の表面が正電荷を帯びるように活性化させることを特徴とする、請求項7に記載のマクロファージの細胞付着・再生を促進する方法。
- 前記マクロファージの細胞付着・再生性分極化を調整するステップは、体内及び体外に前記ナノリガンド提示基板を位置させた後、100〜700mTの磁場を12時間〜48時間印加して行う、請求項7に記載のマクロファージの細胞付着・再生を促進する方法。
- 前記マクロファージの細胞付着・再生性分極化を調整するステップは、基板に印加される磁場の位置を変化させて行う、請求項7に記載のマクロファージの細胞付着・再生を促進する方法。
- 前記マクロファージの細胞付着・再生性分極化を調整するステップは、基板に印加される磁場の位置を経時変化させて行う、請求項7に記載のマクロファージの細胞付着・再生を促進する方法。
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