JP2013535200A - 細胞培養コーティングのためのプレポリマー調製品 - Google Patents
細胞培養コーティングのためのプレポリマー調製品 Download PDFInfo
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- JP2013535200A JP2013535200A JP2013521229A JP2013521229A JP2013535200A JP 2013535200 A JP2013535200 A JP 2013535200A JP 2013521229 A JP2013521229 A JP 2013521229A JP 2013521229 A JP2013521229 A JP 2013521229A JP 2013535200 A JP2013535200 A JP 2013535200A
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- prepolymer
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Abstract
Description
本出願は配列表を含む。配列表に含まれる情報をここに引用する。
式1: Rm−Sp−Cap
により記載される。
工程a:塩化4−ベンゾイルベンゾイルの合成 合成は、塩化ベンゾイル1の公知の合成にしたがった。4−ベンゾイル安息香酸(5g)を100mlの丸底フラスコに入れた。塩化チオニル(15ml)を加えた。この懸濁液を加熱して、1時間に亘り還流し、この最中に全ての固体が溶解した。回転蒸発により、全ての揮発性化合物を除去した。残留物をトルエン中に溶解させ、再び蒸発させた。塩化チオニルを全て除去するために、この操作を二回行った。粗製化合物は、石油エーテルからの再結晶化によりさらに精製できる(溶解度が100ml当たり約1gであることに留意)。この生成物は白色粉末である、Mp.95℃(Lit;:98〜99℃)。1H−NMRは公開データと合致する。1H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 7.5, 2H), 7.86 (d, J = 7.4, 2H), 7.78 (d, J = 7.4, 2H), 7.61 (d, J = 6.7, 1H), 7.49 (t, J = 7.4, 2H) ppm13C NMR (101 MHz, CDCl3) δ 195.31, 167.85, 143.27, 136.36, 135.75, 133.34, 131.15, 130.09, 130.01, 128.59 ppm。=O信号が170.42ppmである炭素スペクトルに、加水分解が容易に見られることに留意。
図2に示したように、撹拌子を備えた琥珀色のフラスコ内の90gのエタノールに、8.0g(0.061ミリモル)のHEMAおよび2.0g(13.9ミリモル)のアクリル酸カルボキシエチル(CEA)を加えた。次いで、0.30g(1.56ミリモル)の2,2’−アゾビス−(2−メチルブチロニトリル)を加え、溶解が完了するまで、撹拌した。1分間に亘りアルゴンをパージしながら、この溶液から酸素を除去した。次いで、密封したフラスコを、混合しなから68℃で24時間に亘り加熱し、光から保護した。室温まで冷却した後、ポリ(HEMA−co−CEA)ポリマーを、酢酸ブチル中での沈殿反応により単離した。得られた白色固体を酢酸ブチルで三回、ジイソプロピルエーテルで三回洗浄した。
これも図2に示されるように、手短に言えば、100mgのポリ(HEMA−co−CEA)を1.5mlの無水DMF中に40℃で混合しながら溶解させた。1.67mlのEDC/NHS(400/200mM)を加え、30分間に亘り撹拌して、ポリ(HEMA−co−CEA)ポリマーのカルボン酸基を活性化させた。これと同時に、2.6mlのホウ酸緩衝液pH9.2および2.90mlのエタノール中に、100mgのビトロネクチンペプチドおよび150μl×Mのローダミン標識ペプチドの溶液を溶解させた。この溶液をDMF中のNHS−活性ポリマーに加え、45分間に亘り撹拌した。次いで、ホウ酸緩衝液pH9.2および1.4mlのエタノール中の2mlの1Mのエタノールアミンからなる溶液を加え、30分間に亘り撹拌した、未反応NHS基をブロッキングした。このポリマーを、公称3500MWCOを有するCell Sup T1再生セルロース管状膜を使用して、透析法により精製した。この透析法は、40%のエタノール/水溶液中で行った。72時間後、溶液をビーカーに移し、0.06ミリバール(6Pa)の圧力下において−89℃で一晩、凍結乾燥させた。結合したポリマーは、エタノール/水混合物、DMSO、DMF、TFE中に非常に可溶性であるピンク色のさらさらした粉末である。
ペプチド(KGGPQVTRGDVFTMP配列番号27)は、以下のプロセスによって、合成され、カリフォルニア州、サニーベール所在のAmerican Peptide社により提供された。(MAA−PEG4−VN):MAA−PEO4−Lys−Gly−Gly−Pro−Gln−Val−Thr−Arg−Gly−Asp−Val−Phe−Thr−Met−Pro−NH2の調製:このペプチドは、Fmoc化学反応により1ミリモルのFmoc−Rink−Amide樹脂上で合成した。アミノ酸に使用した保護基は:AspとThrについてはt−ブチル基、GlnについてはThr基、ArgについてはPbf、LysについてはBocである。Fmoc保護アミノ酸はEMD Biosciences社から購入した;Fmoc−PEG4−OHは、Quanta Biodesign社から購入した。カップリングおよび開裂(cleavage)のための試薬は、Aldrich社から購入した。溶媒はFisher Sceientific社から購入した。ペプチド鎖は、Fmoc保護基のそれぞれの除去および保護されたアミノ酸のカップリングによって、樹脂上に作製した。カップリング剤としてHBTUおよびHOBtを使用し、塩基としてNMMを使用した。脱Fmoc試薬としてDMF中20%のピペリジンを使用した。Fmoc保護基を除去した後、PEG4のアミノ酸にメタクリル酸(MAA)をカップリングさせた。最後のカップリング後、開裂および側鎖の保護基の除去のために、TFA/TIS/H2O(95:3:2、v/v/v)で処理した。粗製ペプチドを冷たいエーテルから沈殿させ、濾過により収集した。収量4.0グラム(合成収率210.1%)。粗製ペプチドを逆相HPLCにより精製した;90%超の純度で収集された分画をプールし、凍結乾燥した。最終生成物の収量1.035g(精製収率25.9%)。
図1に示されるように、撹拌子を備えた琥珀色のフラスコ内の7.5mlのエタノールに、60mg(0.46ミリモル)のHEMA、100mg(0.05ミリモル)のビトロネクチン−PEG4−メタクリレート(通称、VN−PEG4−MAA)を加えた。次いで、9mgの2,2’−アゾビス−(2−メチルブチロニトリル)を加え、溶解が完了するまで、撹拌した。1分間に亘りアルゴンをパージしながら、この溶液から酸素を除去した。次いで、密封したフラスコを、混合しなから68℃で24時間に亘り加熱し、光から保護した。室温まで冷却した後、ポリ(HEMA−co−VNPEGMAA)ポリマーを、酢酸ブチル中に粗製反応媒質を注ぐことにより単離した。得られた白色固体をジイソプロピルエーテルで三回洗浄し、真空下で乾燥させた。下記に記載するコーティングは、ポリ(HEMA−co−VN−PEG4−MAA)ポリマーを精製せずに作製したが、未反応のペプチドは、連続または不連続ダイアフィルトレーションプロセスなどのよく知られたプロセスによって、容易に除去できる。特に効率的な未反応ペプチドの除去は、例えば、5,000 MWCO Corning Spin−X濃縮カラムを使用して、行うことができる。
Photo−HEMAを、図3に示された反応にしたがって調製した。手短に言えば、撹拌子を備えた琥珀色のフラスコ内に秤量した132.8gのエタノールに、12.30g(94ミリモル)のメタクリル酸2−ヒドロキシエチル、HEMAを加え、1.023g(2.9ミリモル)のN−(3−メタクリルアミドプロピル)−4−ベンゾイルベンズアミド(先に記載したプロトコルにしたがって調製した)を加え、この溶液に溶解させた。溶解後、0.48g(2.5ミリモル)の2,2’−アゾビス−(2−メチルブチロニトリル)を加え、溶解が完了するまで、撹拌した。1分間に亘りアルゴンをパージしながら、この溶液から酸素を除去した。次いで、密封したフラスコを、混合しなから68℃で24時間に亘り加熱し、光から保護した。室温まで冷却した後、このポリマーを、酢酸ブチル中での沈殿反応により単離した。得られた固体を酢酸ブチルで三回、ジイソプロピルエーテルで三回洗浄した。次いで、このポリマーを、20μmの多孔質ポリプロピレンフィルタを使用して濾過し、真空下で一晩、乾燥させた。反応工程の全ては、紫外線に直接曝露せずに行われる。
別の実施例において、図3にしたがって、手短に、撹拌子を備えた琥珀色のフラスコ内に秤量した132.8gのエタノールに、12.30g(94ミリモル)のメタクリル酸2−ヒドロキシエチル、HEMAを加え、2.11g(6ミリモル)のN−(3−メタクリルアミドプロピル)−4−ベンゾイルベンズアミド(先に記載したプロトコルにしたがって調製した)を加え、この溶液に溶解させた。溶解後、0.48g(2.5ミリモル)の2,2’−アゾビス−(2−メチルブチロニトリル)を加え、溶解が完了するまで、撹拌した。1分間に亘りアルゴンをパージしながら、この溶液から酸素を除去した。次いで、密封したフラスコを、混合しなから68℃で24時間に亘り加熱し、光から保護した。室温まで冷却した後、このポリマーを、酢酸ブチル中での沈殿反応により単離した。得られた固体を酢酸ブチルで三回、ジイソプロピルエーテルで三回洗浄した。次いで、このポリマーを、20μmの多孔質ポリプロピレンフィルタを使用して濾過し、真空下で一晩、乾燥させた。反応工程の全ては、紫外線に直接曝露せずに行われる。
架橋剤官能化プレポリマーである成分(i)および官能化細胞結合ペプチドプレポリマーである成分(ii)を、トリフルオロエタノール(TFE)中で1から10mg/mlまでの固体含有量で、様々な比率でブレンドすることによって、配合物を調製した。未処理のPS 6W−P Corning Incorporatedプレートを使用した。ウェル当たり26から31μlをアリコートにした(1/4インチ(約6.4mm)の孔のテンプレートの蓋を使用して手作業で)。この蓋をカバープレートと直ちに交換した。適所にカバープレートを配置して、配合物を縁まで広がらせた(相対湿度に応じて5から10分間。空気1kg当たり8〜10gの水が好ましい)。40℃で15分間に亘りカバープレートを濾紙と交換することによって、プレートを乾燥させた(または真空乾燥)。紫外線硬化を、「D」バルブ、80%の出力、0.5のベールと速度、1回通過で行い、プレートは、逆さまにして曝露される(プレートの底面が、ペプチドを保護する紫外線フィルタの役割を果たす)。プレートを1時間に亘りPBSで洗浄し、窒素を流しながら乾燥させた。必要に応じて、プレートをエタノールまたは水中70%v/vのエタノールで洗浄した(消毒を確実にするために)。
Sigma Aldrich社から得られるトリフルオロエタノール(TFE)中の総固体含有量が10mg/mlで、成分(i−b)として、10質量%のPhoto−HEMAおよび成分(ii−b)として、90質量%のポリ(HEMA−co−VNPEGMAA)をブレンドすることによって、コーティング配合物を調製した。各ウェル中に30μlのコーティング溶液を分配することによって、TCT(細胞培養処理)PS 6−WP Corningプレートを被覆した。溶液が完全に広がった後、40℃で15分間に亘りコーティングを乾燥させた。次いで、このプレートを逆さまにして、Fusion「D」バルブ(80%の電力および0.6m/分のベルト速度)により提供される光に曝露することによって、コーティングを紫外線架橋させた。次いで、プレートを1時間に亘り1%のドデシル硫酸ナトリウム水溶液で洗浄し、脱イオン水で完全に濯ぎ、最後に、穏やかに乾燥ガス流を流して乾燥させた。このコーティングはDH2105−10Aと呼ばれる(無血清ESC培養データに関する図12C参照);BCAアッセイにより決定された固定化ペプチド密度が、固定化ペプチド密度が約30〜35ピコモル/mm2であることを示した。細胞培養の前に、プレートを、70%v/vのエタノール水溶液を加えることにより消毒したか、またはガンマ線への曝露により滅菌した(本発明のコーティング組成物は、細胞接着性能を損なわずに、ガンマ線滅菌が可能であることは注目に値する)。
HT1080細胞(ATCC#CCL−121)を、10%のFBSおよびペニシリンストレプトマイシンが補われた培地である、カリフォルニア州、カールズバッド所在のLife Technologies社のイスコフ改変ダルベッコ培地中で維持した。細胞をトリプシン処理し、飽和密度に到達する前に希釈した。
Claims (5)
- 高分子細胞培養表面を形成するための組成物であって、第1の高分子主鎖に結合した細胞接着性ペプチドを含む第1のプレポリマー、および第2の高分子主鎖に結合した架橋剤部分を含む第2のプレポリマーを有してなり、
前記第1のプレポリマーの細胞接着性ペプチドが式Rm−Sp−Capを有し、
式中、
Rは、アクリレート、メタクリレート、アクリルアミド、メタクリルアミド、マレイミドおよびフマレート並びにそれらの任意の組合せからなる群より選択される重合部分であり、mは1より大きい整数であり、
Spは、R’がHまたはCH3であり、m2が0から20までの整数である式(O−CH2CHR’)m2により表されるポリエチレンオキシドまたはポリプロピレンオキシド;またはXaaが独立して任意のアミノ酸であり、nが0から20までであるXaan;もしくはそれらの任意の組合せを含む随意的なスペーサ部分であり、
Capは、細胞接着性配列を有するペプチドであり、
前記第2のプレポリマーの前記第2の高分子主鎖が、エチレン性不飽和モノマーの重合生成物を含む、組成物。 - 前記第1のプレポリマーの前記第1の高分子主鎖が、カルボキシル基を有するエチレン性不飽和モノマーと、カルボキシル基を有さないエチレン性不飽和モノマーとの重合生成物を含み、前記細胞接着性ペプチドが前記カルボキシル基を通じて前記第1のプレポリマーに結合している、請求項1記載の組成物。
- 前記第1のプレポリマーのエチレン性不飽和モノマーが親水性である、請求項1または2記載の組成物。
- 前記第2のプレポリマーのエチレン性不飽和モノマーが親水性である、請求項1から3いずれか1項記載の組成物。
- 前記第2のプレポリマーが、ポリHEMAに結合したベンゾフェノン架橋剤部分を含む、請求項1から4いずれか1項記載の組成物。
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