【作者】 李林辉；

【导师】 高长有；

【作者基本信息】 浙江大学 ， 材料学， 2009， 博士

【摘要】 生物材料(biomaterial)能够以一种安全、可靠、经济且生理相容的方式在结构或功能上代替身体部分组织或器官的功能。硅基材料在生物医学和生物技术的发展中扮演着重要的角色，而改善硅基材料表面的生物相容性非常重要。我们利用表面引发的原子转移自由基聚合(ATRP)，将温敏聚合物聚(N-异丙基丙烯酰胺)(PNIPAAm)接枝到硅片表面，制备了温敏的细胞培养表面，研究了温敏表面对细胞粘附和温敏分离的调控。通过表面引发的原子转移自由基聚合，在硅片表面接枝了PNIPAAm聚合物膜，并用原子力显微镜、椭偏仪、X射线光电子能谱、接触角、石英晶体微天平等对表面进行了表征。结果表明，PNIPAAm成功地接枝到了硅片表面，PNIPAAm分子量即表面聚合物膜的厚度具有很好的可控性，且表面具有温度敏感性。利用注射法结合表面引发原子转移自由基聚合，制备了PNIPAAm的厚度梯度，研究了PNIPAAm刷厚度对细胞粘附和温敏分离的影响。结果发现，在PNIPAAm接枝表面，适于细胞粘附和温敏分离的厚度为20-45nm。聚乙二醇(PEG)分子的引入可以促进PNIPAAm链的水化作用。利用ATRP聚合在硅片表面接枝了一层PEG分子，再以PEG为大分子引发剂引发NIPAAm聚合，得到P(PEGMA)-b-PNIPAAm的嵌段聚合物刷。与注射法相结合，得到不同梯度走向的三种共聚物梯度表面，发现PEG大分子的引入，使表面在温度降低时的水化速度加快，从而促进了细胞从表面分离。为了改善PNIPAAm表面的细胞粘附，我们在表面固定了RGD肽。首先用表面引发原子自由基聚合接枝了PNIPAAm刷，再以PNIPAAm的活性端基为引发剂，通过丙烯酸钠的ATRP聚合接枝聚丙烯酸(PAA)，再通过羧基与氨基之间的官能团偶合，将RGD肽固定到了温敏表面。利用梯度法研究了RGD接枝量对细胞粘附和温敏分离的影响。结果发现，随PAA接枝量增大，RGD的含量增加，细胞在表面的粘附增多，RGD的表面固定促进了细胞在温敏表面的粘附。本研究为硅基温敏表面在生物医学及材料学上的研究及应用提供了理论和实践基础。更多还原

【Abstract】 Biomaterial science is an important interdisciplinary subject derived frommaterial science and life science.Biomaterials or biomedical materials couldsubstitute the function of a part of tissues or organics through a safe,reliable,biocompatible way.The Thermoresponsive poly（N-isopropylacrylamide） （PNIPAAm）has a lower critical solution termperature （LCST） at about 32℃in pure water.It isuseful for cell recovery without enzymatic digestion.Cells can adhere,spread,andproliferate on the surface grafted with PNIPAAm at 37℃,but can be spontaneouslydetached by only lowering the culture temperature to below the LCST of PNIPAAm.Here,we fabricated thermoresponsive cell culture surfaces using surface-initiatedatom transfer radical polymerization （SI-ATRP） of NIPAAm on the silicon substratesand then studied the manipulation of the surface on cell adhesion and detachment.Firstly,3-（aminopropyl）triethoxysilane （APS） was immobilized on the siliconsurface to introduce-NH₂ groups on the surface.Then the substrate was treated withATRP initiator,bromoisobutyryl bromide,to give rise to the Si-Br surface.TheSI-ATRP of NIPAAm on the Si-Br surface was carried out using a reaction mixture ofNIPAAm,CuBr,PMDETA （1,1,4,7,7-pentamethyldiethylenetriamine）,H₂O/MeOH.After polymerization,the PNIPAAm grafted surfaces were characterized by atomicforce spectroscopy （AFM）,ellipsometry,X-ray photoelectron spectroscopy （XPS）,contact angle,quartz crystal microbalance （QCM）,respectively.The experimentalresults showed that PNIPAAm chains had been successfully grafted on the siliconsurfaces,the chains of PNIPAAm grew linearly from the silicon substrates,and thePNIPAAm surfaces showed temperature sensibility.At 37℃,cells adhere,proliferate on the PNIPAAm surfaces,while lowering the culture temperature to theLCST of PNIPAAm,cells detached spontaneously from the surfaces.To study the effect of the thickness of PNIPAAm grafted on the silicon surface oncell adhesion and detachment,the thickness gradient surface has been prepared usinga combinatorial method:a micropump was used to control the polymerization time ofdifferent positions on the silicon substrate.The results showed that the surface became more hydrophilic with the increase of the thickness of PNIPAAm.When the thicknessof PNIPAAm was higher than 45nm,cells could not adhere on the surface.Lowerthan 20nm,the cells could not detach from the surface as lowering culturetemperature to 20℃.So the suitable thickness for cell adhesion and thermoresponsivedetachment of PNIPAAm on the silicon surface was about 20-45nm.Introducing poly（ethylene glycol） （PEG） molecules into PNIPAAm chains couldaccelerate the hydration of PNIPAAm chains.So a layer of PEG was firstlyimmobilized on the silicon surface by the ATRP of poly（ethylene glycol） methylmethacrylate （PEGMA）,then PNIPAAm chains were formed from the PEG surfaceusing P（PEGMA） as macromolecular initiators.Thus block copolymer brushes ofP（PEGMA）-b-PNIPAAm were achieved.Combined with the injecting method,threetypes of gradient surfaces with different gradient direction were fabricated on thesilicon substrates.Cell culture results showed that the introduction of PEG moleculesmade the PNIPAAm chains hydrate quickly during the process of lowering the culturetemperature,thus the cells could detach quickly from the PNIPAAm surfaces.To improve cell adhesion on the PNIPAAm grafted surface,Arg-Gly-Asp （RGD）peptide was gradiently immobilized on the thermoresponsive surface.Firstly,PNIPAAm brushes were grafted on the silicon surface by the SI-ATRP of NIPAAm,then polyacrylic acid （PAA） chains grew from the PNIPAAm surface through theATRP of sodium acrylate.Finally,the RGD peptide was covalently grafted on thesurface by the functional group reaction of carboxyl group and amino group.Theeffect of PAA grafting thickness and RGD grafting quantity on cell adhesion anddetachment of thermoresponsive surface was studied.With the increase of PAAthickness,grafting quantity of RGD increased and so the cells adhered on the surface.But when the PAA thickness increased to some content,the negative effect ofcarboxyl groups on cell adhesion appeared,because the surface became morehydrophilic and thereby the number of cells adhered on the surface decreased.更多还原