Development of bioresponsive material for controllong cell proliferation and exfoliation

开发用于控制长期细胞增殖和脱落的生物响应材料

• 批准号: 15500334
• 负责人: SUGIYAMA Kazuo
• 金额: $ 1.92万
• 依托单位: Kinki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15500334/
• 关键词: 2-hydroxypropyl methacrylamide; methyl methacrylate; Sodium Copper-chlorophyllin; thermoresponsible polymer; light-responsible polymer; cell exfoliation; HeLa Cell; 3T3 Cell; 2-hydorxypropylmethacrylamlde; 2-hydroxypropylmethacrylamide

Novel thermoresponsible random copolymers, poly(HPMA-co-MMA), and light responsible random copolymers, poly(HPMA-co-MMA/Chlph) were prepared from biocompatible 2-hydroxypropylmethacrylamide(HPMA), methyl methacrylate (MMA), and sodium copper-chlorophyllin(Chlph) as a basic polymer backbone to give an artificial extra cellular matrix for controlling cell proliferation and exfoliation. This research results ranged from (I) to (V) as described below in basic and application examination.(I)Graft-polymerization onto diamond like carbon (DLC) coating material : The graft-polymerization of vinyl monomer was developed onto DLC coated inorganic materials on three-dimensional pieces with complex shapes based on low temperature plasma irradiation/post-polymerization technique onto polymer material.(II)Development of novel polymer material as an artificial extra cellular matrix : RGDS(cell attachment peptide) was immobilized on the thermoresponsible poly(HPMA-co-MMA) grafted polyethyleneterephthal … More ate(g-PET) as a substrate via two step reaction as follows : (1)N-methacryloylsuccinimide (NASu) was grafted onto g-PET by Ar plasma / post polymerization, (2)Synthetic RGDS was reacted with MASu grafted g-PET. The rate of HeLa cells proliferation until 48 hr was considerably increased comparing to non-RGDS treated poly(HPMA-co-MMA) under normal cell cultivative condition. The more than 80% of cultured HeLa cells were exfoliated by decreasing temperature (25℃) for 2hr while non RGDS treated poly(HPMA-co-MMA) and untreated PET was about 70% and 10%, respectively Platelets adhesion was suppressed on newly designed a zwitterionic poly(N^α-methacrylamide-L-lysiene) grafted film as a typical example among examined samples. It was found that the developed polymer surface modification enables us to control cell attachment and exfoliation. The evaluation of surface properties for designing appropriate chemical structure was newly proposed by using bacterial random peptide library method(FliTrx^<TM> : Invitrogen). The hydrophilic-hydrophobic change of poly(HPMA-co-MMA) grafted PET on heating was able to be shown by using hydrophobic parameters from selective amino acid sequences.(III)Light responsible polymer material as a cell scaffold : Poly(HPMA-co-MMA/Chlph) showed a lower critical solution temperature(LCST) under visible light irradiation. For example, the maximum 68% of attached 3T3 cells was exfoliated after turning off the light irradiation for 2hr. The novel cell culture and exfoliation systems was newly developed by visible light controlling.(IV)Microporous hydroxyapatide(HAP) and/or visible light responsible TiO_2 hybrid material : HAP and/or TiO_2 hybrid material as a scaffold was prepared as follows ; (1)Microspheres with phosphoric acid on the surface was allowed to stand at 80 ℃ in HAP forming and/or TiO_2 suspended solution to give HAP/TiO_2 coated microspheres(HAP/TiO_2 -MS). (2)The molded HAP/TiO_2-MS to like disk was calcinated at 600℃ to give a microprous HAP/TiO_2.The cell attachment and exfoliation is now under consideration.(V)Thermo- and/or pH sensitive hydrogels : Molecularly imprinted thermosenstive and/or pH responsible hydrogels obtained from N-isopropylacrylamide and/or zwitterionic O-methacryloyl-L-serine were prepared far basic examinations for controlling and timing release of such as cell differentiation or proliferation factors. Less

以2-羟丙基甲基丙烯酰胺（HPMA）、甲基丙烯酸甲酯（MMA）和叶绿酸铜钠（Chlph）为基本聚合物骨架，制备了新型热响应性无规共聚物（Poly（HPMA-co-MMA））和光响应性无规共聚物（Poly（HPMA-co-MMA/Chlph）），得到了用于控制细胞增殖和脱落的人工细胞外基质。本研究结果在基础和应用测试中的范围从（I）到（V）如下所述。（I）类金刚石碳（DLC）涂层材料的接枝聚合：基于聚合物材料的低温等离子体辐照/后聚合技术，开发了乙烯基单体在具有复杂形状的三维片上的DLC涂层无机材料上的接枝聚合。（II）开发新型高分子材料作为人工细胞外基质：将RGDS（细胞附着肽）固定在热响应性聚（HPMA-co-MMA）接枝的聚对苯二甲酸乙二醇酯上 ...更多信息 （1）通过Ar等离子体/后聚合将N-甲基丙烯酰琥珀酰亚胺（NASu）接枝到g-PET上;（2）合成RGDS与MASu接枝的g-PET反应。在正常细胞培养条件下，与未经RGDS处理的聚（HPMA-co-MMA）相比，HeLa细胞增殖速率显著增加，直至48小时。结果表明，经25℃降温处理2小时，HeLa细胞脱落率达80%以上，而未经RGDS处理的聚（HPMA-co-MMA）和未经处理的PET细胞脱落率分别为70%和10%。结果发现，开发的聚合物表面改性使我们能够控制细胞附着和脱落。通过使用细菌随机肽文库方法（FliTrx ®：Invitrogen）新提出了用于设计适当化学结构的表面性质的评价<TM>。聚（HPMA-co-MMA）接枝PET在加热时的疏水-疏水变化能够通过使用选择性氨基酸序列的疏水参数来显示。（III）作为细胞支架的光响应性聚合物材料：聚（HPMA-co-MMA/Chlph）在可见光照射下显示出较低的临界溶解温度（LCST）。例如，在关闭光照射2小时后，最多68%的附着的3 T3细胞脱落。利用可见光调控技术，开发了一种新型的细胞培养和脱落系统。（4）多孔羟基磷灰石（HAP）和/或可见光响应性TiO_2杂化材料：HAP和/或TiO_2杂化材料作为支架材料的制备方法如下：（1）将表面带有磷酸的微球置于HAP形成液和/或TiO_2悬浮液中，于80 ℃静置，得到HAP/TiO_2包覆微球（HAP/TiO_2 -MS）。（2）将HAP/TiO_2-MS成型为片状，600℃煅烧，得到微多孔的HAP/TiO_2。（V）热敏性和/或pH敏感性水凝胶：从N-异丙基丙烯酰胺和/或两性离子O-甲基丙烯酰基-L-丝氨酸获得的分子印迹热敏性和/或pH响应性水凝胶被制备用于控制和定时释放例如细胞分化或增殖因子的基本检查。少

项目成果

医療用材料及びそれを用いた医療用材料の製造方法

医用材料以及使用其制造医用材料的方法

• 发表时间: 2004

Manufacturing method of Biommaterial for medical material and medical equipment application

医用材料及医疗器械应用的生物材料制造方法

• 期刊: Japanese patent (2004-100186) PCT/JP2005/005534
• 作者: Akihiro Kanematsu;Akira Marui;Shingo Yamamoto;Makoto Ozeki;Yoshiaki Hirano;Masaya Yamamoto;Osamu Ogawa;Masashi Komeda;Yasuhiko Tabata;Kazuo Sugiyama
• 通讯作者: Kazuo Sugiyama

温度応答性ポリ(2-ヒドロキシプロピルメタクリルアミド)グラフト化PET表面の大腸菌ランダムペプチドライブラリー法による評価

使用大肠杆菌随机肽库方法评估温度响应型聚（2-羟丙基甲基丙烯酰胺）接枝 PET 表面

• 发表时间: 2005
• 期刊: 高分子学会予稿集 54巻(発表予定)
• 作者: 杉山一男;他4名
• 通讯作者: 他4名

Kazuo Sugiyama, 他2名: "Characterization and application of polypropylene films modified with stimuli-sensitive copolymers with an Ar-plasma postpolymerization technique"Journal of Applied Polymer Science. 90巻. 143-148 (2003)

Kazuo Sugiyama 等 2 人：“采用 Ar 等离子体后聚合技术对刺激敏感共聚物进行改性的聚丙烯薄膜的表征和应用”，应用聚合物科学杂志，第 90 卷，143-148（2003 年）。

杉山一男, 他3名: "クロロフィリン銅錯体を含む2-ヒドロキシプロピルメタクリルアミド/MMAコポリマーの光・熱刺激応答と細胞増殖用基材への応用"高分子学会予稿集. 52巻・5号. 1085 (2003)

Kazuo Sugiyama 等 3 人：“含叶绿素铜复合物的 2-羟丙基甲基丙烯酰胺/MMA 共聚物对光和热刺激的响应及其在细胞增殖基质中的应用”，高分子科学技术学会论文集，第 52 卷。 5. 1085 (2003)