Development of bioresponsive material for controllong cell proliferation and exfoliation

以具有生物相容性的2-羟丙基甲基丙烯酰胺（HPMA）、甲基丙烯酸甲酯（MMA）和叶绿素铜钠（Chlph）为基本聚合物骨架，制备了新型热负责无规共聚物聚（HPMA-co-MMA）和光负责无规共聚物聚（HPMA-co-MMA/Chlph），为控制细胞增殖和脱落提供了一种人工细胞外基质。本研究结果在基础及应用考试中所述范围为(I)至(V)。（一）DLC涂层材料接枝聚合：基于低温等离子体辐照/高分子材料后聚合技术，在三维复杂形状的DLC涂层无机材料上开展了乙烯基单体接枝聚合。(2)将RGDS（细胞附着肽）固定在热负责型聚（HPMA-co-MMA）接枝的聚乙二醇上，通过Ar等离子体/后聚合将n -甲基丙烯酰丁二酰亚胺（NASu）接枝到g-PET上，通过两步反应将合成RGDS与MASu接枝的g-PET反应。在正常细胞培养条件下，与非rgds处理的poly（HPMA-co-MMA）相比，HeLa细胞增殖至48小时的速率显著增加。降温（25℃）2hr后，80%以上的HeLa细胞脱落，未RGDS处理的聚（HPMA-co-MMA）和未RGDS处理的聚（PET）分别约为70%和10%，新设计的两性离子聚（N^α-甲基丙烯酰胺-l -赖烯）接枝膜的血小板粘附被抑制为典型。发现所开发的聚合物表面修饰使我们能够控制细胞附着和脱落。采用细菌随机肽库法（FliTrx^<TM>: Invitrogen）评价表面性质，设计合适的化学结构。利用选择性氨基酸序列的疏水参数，可以表征聚（HPMA-co-MMA）接枝PET在加热过程中的亲疏水变化。（三）作为细胞支架的光负责高分子材料：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，在关闭光照2小时后，附着的3T3细胞脱落率达到68%。利用可见光控制技术开发了一种新的细胞培养和剥离系统。(4)微孔羟基磷灰石（HAP）和/或TiO_2杂化材料：制备HAP和/或TiO_2杂化材料作为支架；(1)表面有磷酸的微球在HAP形成和/或TiO_2悬浮液中放置80℃，得到HAP/TiO_2包覆微球（HAP/TiO_2 -MS）。(2)将成型的HAP/TiO_2- ms在600℃下煅烧，得到微孔HAP/TiO_2。现在正在考虑细胞附着和脱落。(V)热敏和/或pH敏感水凝胶：由n -异丙基丙烯酰胺和/或两性离子o-甲基丙烯酰- l-丝氨酸制备的分子印迹热敏和/或pH敏感水凝胶用于控制和定时释放细胞分化或增殖因子的基础检查。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