JP5673596B2 - 細胞膜透過性を有する製剤及び細胞膜透過性組成物 - Google Patents
細胞膜透過性を有する製剤及び細胞膜透過性組成物 Download PDFInfo
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Description
前記化合物がオリゴアルギニン構造の末端にパルミチン酸が結合した化合物である製剤に関する。
ヒト血清アルブミン(HSA)として、献血アルブミン20”化血研”(化学及血清療法研究所)を用いた。このHSA溶液(20質量%)を、塩酸を用いてpH3に調整した。HSAの質量(HSA乾燥重量)に対して半量の活性炭を加え、1時間撹拌した。20000gで20分間遠心分離することによって活性炭を取り除いた後、NaOH水溶液を用いてpH7.4に調整した。その後、透析して脱塩処理し、さらに凍結乾燥することで、脱脂処理HSAを調製した。
4mg/mLの脱脂処理したHSA(溶媒は0.15M K2HPO4である)1.0mLに対して1mg/mL FITC(溶媒は0.15M K2HPO4である)0.5mLを混合し、常温、遮光条件下で4時間インキュベートした。その後、混合液をG-25ゲルろ過カラムに流し、PBSを用いてFITC標識HSAを溶出させた。蛋白質濃縮キット(ザルトリウス社VIVAPORE)を用いて溶出液中の蛋白質(FITC標識HSA)を100〜200μLとなるまで濃縮した。
<3.化学式1の化合物の製造>
ペプチド合成機として、島津社製PSSM-8を使用した。最初に樹脂(渡辺化学Fmoc-NH-SAL-resin)をDMF/20%ピペリジンを用いて脱Fmocした。DMFを用いて樹脂を洗浄した後、縮合剤カクテル(HBTU+HOBt・H2O+DMF)及びDIEA存在下でペプチド配列C末端一番目のFmocアミノ酸をカップリング反応により樹脂に結合させた。DMFを用いた樹脂の洗浄を挟み、(脱保護→カップリング反応)というステップを繰り返し行い、(Arg)12というペプチドを得た。さらに、脱Fmoc後にパルミチン酸をカップリング反応により結合させることによって、化学式1の化合物を製造した。
PBSに溶解させた30μg/mLのFITC標識HSA溶液を、化学式1の化合物溶液(溶媒PBS、濃度0.25mg/mL)と1:1のモル比(HSAと化合物のモル比)で混合した。この溶液を実施例1の製剤とした。
<5.化学式2の化合物の製造>
実施例1と同様にして、脂肪酸-Gly-Ser-Ser-Gly-Cys-(Arg)12-Cysという化合物を得た。この化合物をPBS中で分子内ジスルフィド結合させることにより、化学式2の化合物を製造した。
PBSに溶解させた30μg/mLのFITC標識HSA溶液を、化学式2の化合物溶液(溶媒PBS、濃度0.25mg/mL)と1:1のモル比(HSAと化合物のモル比)で混合した。この溶液を実施例2の製剤とした。
HeLa細胞を24wellsプレートに7×104cellsとなるように播種した。細胞の培地として、D-MEM(+10%FBS)培地を使用した。播種後、24wellsプレートを恒温槽に保管し、37℃で24時間インキュベートした。上清を除去後、D-MEM培地(FBSなし)に培地交換し、3時間スタベーションを行った。スタベーション後、実施例1又は実施例2の製剤50μLを各wellに添加した。24wellsプレートを恒温槽に保管し、37℃で2時間インキュベートした。
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