Development of bioresponsive material for controllong cell proliferation and exfoliation

以生物相容的甲基丙烯酰胺(HPMA)、甲基丙烯酸甲酯(MMA)和叶绿素钠(Chlph)为基本聚合物骨架，制备了新型热敏无规共聚物聚(HPMA-co-MMA)和光敏感无规共聚物聚(HPMA-co-MMA/Chlph)，为控制细胞增殖和脱落提供了人工细胞外基质。(I)类金刚石涂层材料的接枝聚合：基于聚合物材料上的低温等离子体辐照/后聚合技术，将乙烯基单体接枝聚合到三维形状复杂的类金刚石涂层无机材料上。(Ii)新型聚合物材料的开发作为人工细胞外基质：将细胞附着肽固定在热响应性聚合物(HPMA-co-MMA)接枝的聚乙烯对苯二甲酸乙二醇酯(…)上(1)N-甲基丙烯酰基琥珀酰亚胺(NSAU)通过Ar等离子体/后聚合接枝到g-PET上；(2)合成的RGDS与MASU接枝g-PET反应。在正常细胞培养条件下，HeLa细胞48小时的增殖率明显高于未经RGDS处理的聚(HPMA-co-MMA)。体外培养的HeLa细胞经降温(25℃)2小时后，80%以上的细胞被剥离，而未经RGDS处理的聚合物(HPMA-co-MMA)和未经处理的聚合物分别约为70%和10%。在所考察的样品中，以新型两性离子聚(N^α-甲基丙烯酰胺-L-赖氨酸)接枝膜为例，血小板黏附受到抑制。研究发现，所开发的聚合物表面修饰使我们能够控制细胞的附着和剥落。利用细菌随机肽库方法(FliTrx^&lt；TM&gt；：Invitgen)对表面性质进行评价，为设计合适的化学结构提供了新的方法。从氨基酸序列的疏水参数可以看出聚(HPMA-co-MMA)接枝PET在加热过程中亲水性-疏水性的变化。(Iii)作为细胞支架的光响应性聚合物材料：Poly(HPMA-co-MMA/Chlph)在可见光照射下表现出较低的临界溶解温度(LCST)。例如，关闭光照射2小时后，附着的3T3细胞最大有68%被剥离。(4)制备了微孔羟基磷灰石和/或可见光响应型二氧化钛杂化材料：以羟基磷灰石和/或二氧化钛杂化材料为支架：(1)在羟基磷灰石形成和/或二氧化钛悬浮液中放置80℃，制得羟基磷灰石/二氧化钛包覆微球(HAP/TiO2-MS)。(2)模制的羟基磷灰石/二氧化钛-微球在600℃下焙烧，得到微孔羟基磷灰石/二氧化钛。(5)温敏和/或pH敏感水凝胶：从N-异丙基丙烯酰胺和/或两性离子O-甲基丙烯酰基-L-丝氨酸制备的分子印迹温敏和/或pH响应性水凝胶是控制和定时释放细胞分化或增殖因子的重要基础检查。较少

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