【作者】 涂琴；

【导师】 王进义；

【作者基本信息】 西北农林科技大学 ， 化学生物学， 2013， 博士

【摘要】 作为一多学科交叉的研究方向，功能聚合物界面的构建及其生物学应用越来越受到人们的关注，并已成为目前生命分析领域的研究热点，其主要原因是聚合物的性质可以通过不同的化学反应进行人为的调节和控制，从而构建适宜于不同研究对象和目的功能界面。目前，已经有许多聚合物界面被用于不同的生物学应用。然而，由于目前所用材料固有的一些缺点（如表面惰性而无法功能化，对生物样品的非特异性吸附等）严重制约了这些聚合物界面在生命分析中的进一步应用。研究发现新型聚合物材料的合成及其功能界面的构建是解决该问题的关键。鉴于此，本研究的主要内容是设计、合成具有一定生物功能的聚合物，并以其为基底，根据不同的研究目的构建其生物学界面，同时对所构建界面在生命分析中的应用进行探索。通过上述研究工作，取得了以下重要结论：1.通过点击化学和自由基聚合反应合成了一类新型的乙酰胆碱类仿生聚合物。合成的聚合物包括生物活性单元（乙酰胆碱类似物单元）和生物惰性单元（聚乙二醇单元）。为了进一步探索生物活性单元和生物惰性单元对神经元生长的影响，通过改变这两种单体的比例，合成5种不同的聚合物，并通过氢核磁共振谱（1H nuclear magnetic resonance,1H NMR）、傅里叶红外谱（Fourier-transform infrared spectroscopy, FT-IR）、凝胶渗透色谱（Gel permeation chromatography, GPC）和差示扫描量热仪（Differential scanningcalorimetry, DSC）测定了其物理性质。通过在这些聚合物所构建的膜界面上培养鼠海马神经元研究发现：调节聚合单体的比例会明显影响神经元的生长；鼠神经元在不同的聚合物表面显示出了不同的生长形态；在生物惰性单元与生物活性单元比例为1:60时，制备的聚合物膜表面可诱导神经元的再生，其能力类似于聚赖氨酸（poly-L-lysine, PLL）（一种培养神经细胞的常用基底材料）。这些乙酰胆碱类仿生聚合物可作为仿生材料在神经工程中具有一定的应用前景，特别是在调节海马神经元生长方面的应用。2.通过水相氧化反应和甲基丙烯酸二甲氨乙酯（Dimethylaminoethyl methacrylate,DMAEMA）为单体的表面自由基聚合（Surface-initiated atom transfer radical polymeri-zation, SI-ATRP）反应，成功制备了一种季铵化聚甲基丙烯酸二甲氨乙酯[Quaternizedpoly(dimethylaminoethyl methacrylate), QPDMAEMA]嫁接的聚二甲基硅氧烷[Poly(dimethylsiloxane)，PDMS]表面。其步骤如下：首先在H2SO4/H2O2溶液中，将PDMS表面的Si-CH3转换为Si-OH。然后，通过硅烷化反应将引发剂固定在其表面，并通过原子转移自由基聚合将聚甲基丙烯酸二甲氨乙酯（PDMAEMA）接枝到PDMS表面。最后通过溴乙烷与PDMAEMA上的亚胺结合形成季铵化的PDMAEMA刷修饰的PDMS表面。采用全反射红外光谱（Attenuated total reflection FT-IR,ATR-FT-IR）、X射线光电子能谱（X-ray photoelectron spectroscopy, XPS）、接触角测定等手段，对所制备的季铵化PDMAEMA修饰PDMS界面进行了表征与分析。研究结果显示，与没有修饰的PDMS表面以及PDMAEMA修饰的PDMS表面相比，季铵化的PDMAEMA修饰PDMS表面具有很好的湿润性、稳定性，同时具有显著的抗蛋白吸附、抗细菌粘附和细胞粘附的能力。3.通过单甲氧基聚乙二醇引发的乳酸开环聚合及随后点击化学嫁接含叠氮基的聚甲基丙烯酸磺基甜菜碱的反应，合成了一类新型的抗生物粘附的聚酯，即：聚乙二醇-聚乳酸-聚磺基甜菜碱[monomethoxy-poly(ethylene glycol)-b-poly(L-lactide)-b-poly(sulfobetaine methacrylate), MPEG-PLA-PSBMA]。聚合物的化学结构采用氢核磁共振谱（1H NMR）和傅里叶红外谱（FT-IR）进行了表征，其物理性质（如分子量、玻璃化温度和熔点）通过凝胶渗透色谱（GPC）和差示扫描量热仪（DSC）进行了测定。为了探索此类聚合物的亲水性及其抗生物粘附性能，采用物理涂层的方法制备了系列此类聚合物的膜表面，并通过接触角的测定研究了其亲水性和稳定性，通过不同蛋白的吸附、不同细胞和细菌的培养研究了其抗生物粘附能力。结果显示制备的两性的聚酯具有很好的可湿性和稳定性，以及优良的抗生物粘附能力。4.由于其无毒、无过敏和良好的生物相容性，脂肪族聚酯在功能生物材料中应用与研究引起了广泛的关注。但是由于脂肪族聚酯的疏水性和侧链功能基团的缺乏严重限制了其在生物医学领域的应用。为此，该研究也通过带末端炔基内酯的开环聚合及随后的点击化学嫁接合成了四种新型的具有不同功能基团的脂肪族聚酯：聚甲基丙烯酸磺基甜菜碱（polySBMA-）、聚甲基丙烯酸乙基磷酸胆碱[poly(2-methacryloyloxylethylphosphorylcholine), polyMPC-]、聚乙二醇[poly(ethylene glycol), PEG-]和季铵化的聚甲基丙烯酸二甲氨乙酯（QPDMAEMA-）嫁接的聚丙炔已交酯-聚已内酯[poly(propargylglycolide)-co-poly(ε-caprolactone), PPGL-co-PCL]。聚合物的化学结构通过氢核磁共振谱（1H NMR）和傅里叶红外谱（FT-IR）进行了表征，其物理性质（如分子量、玻璃化温度和熔点）通过凝胶渗透色谱（GPC）和差示扫描量热仪（DSC）进行了测定。为了探索这些聚合物的亲水性和抗生物粘附的性能，采用物理涂层的方法制备了系列这些聚合物膜表面，并对其亲水性、稳定性及抗蛋白、细胞及细菌粘附的能力进行了评价。结果显示，所制备的功能化脂肪族聚酯具有很好的亲水性、稳定性及抗蛋白、细胞粘附的性能，同时也具有抑制细菌粘附和生长的能力。综上所述，该研究成功合成了系列具有不同生物功能的聚合物，构建了系列聚合物表面，并初步探索了其在生命分析中的应用。该研究对于不同生物功能材料制备的方法与技术、不同生物功能材料的性质及其生物学应用进行了系统深入的研究。同时，对这些聚合物在生物医学领域的进一步应用进行了分析与讨论。更多还原

【Abstract】 As a multi-disciplinary research direction, constructing the function interface haspenetrated in various field. Especially the interface sensing and its applications have attractedmore and more attention. Among construct functional polymer interface and biologicalapplications is the focus of current research. Mainly due to the properties of the polymer canbe artificial adjusted by different chemical reactions for the different objects. At present,many polymers have been used to construct functional interfaces for different biologicalapplications. However, the inherent shortcomings of these materials, such as surface inert, theviability of interface cell and nonspecific adsorption, serious constraint their furtherapplications in life analysis field. We found that synthesizing the new materials andconstructing the function interface was the key to solve this problem. Therefore, the maincontent of the present study was preparation of functional polymers and their applications inbiology. According to our present results, the conclusions were made as follows.1. In this study, a new type of acetylcholine-like biomimetic polymers for their potentialin biomaterial-modulated nerve regeneration application is synthesized using click chemistryand free radical polymerization. The structure of the synthesized polymers includes a“bioactive” unit (acetylcholine-like unit) and a “bioinert” unit [poly(ethylene glycol) unit]. Toexplore the effects of the bioactive unit and the bioinert unit on neuronal growth, differentratios of the two initial monomers poly(ethylene glycol) monomethyl ether-glycidylmethacrylate (MePEG-GMA) and dimethylaminoethyl methacrylate (DMAEMA) wereemployed and five different polymers were synthesized. Their chemical structures werecharacterized using1H nuclear magnetic resonance (1H NMR) and Fourier-transform infraredspectroscopy (FT-IR), and their physical properties (including molecular weight,polydispersity, glass transition temperature, and melting point) were determined using gelpermeation chromatography (GPC) and differential scanning calorimetry (DSC). Culturing ofthe primary rat hippocampal neurons on the polymeric surfaces show that the ratio of the twoinitial monomers utilized for polymer synthesis significantly affects neuronal growth. Rathippocampal neurons show different growth morphologies on different polymeric surfaces.The polymeric surface prepared with1:60(mol/mol) of MePEG-GMA to DMAEMA induces neuronal regenerative responses similar to that on poly-L-lysine, a very common benchmarkmaterial for nerve cell cultures. These results suggest that acetylcholine-like biomimeticpolymers are potential biomaterials for neural engineering applications, particularly inmodulating the growth of hippocampal neurons.2. A quaternized poly(dimethylaminoethyl methacrylate)-grafted poly(dimethylsiloxane)(PDMS) surface (PDMS-QPDMAEMA) was successfully prepared in this study viasolution-phase oxidation reaction and surface-initiated atom transfer radical polymerization(SI-ATRP) using dimethylaminoethyl methacrylate (DMAEMA) as initial monomer. PDMSsubstrates were first oxidized in H2SO4/H2O2solution to transform the Si-CH3groups on theirsurfaces into Si-OH groups. Subsequently, a surface initiator for ATRP was immobilized ontothe PDMS surface, and DMAEMA was then grafted onto the PDMS surface viacopper-mediated ATRP. Finally, the tertiary amino groups of PolyDMAEMA (PDMAEMA)were quaternized by ethyl bromide to provide a cationic polymer brush-modified PDMSsurface. Various characterization techniques, including contact angle measurements,attenuated total reflection infrared spectroscopy (ATR-FT-IR), and X-ray photoelectronspectroscopy (XPS), were used to ascertain the successful grafting of the quaternizedPDMAEMA brush onto the PDMS surface. Furthermore, the wettability and stability of thePDMS-QPDMAEMA surface were examined by contact angle measurements. Antifoulingproperties were investigated via protein adsorption, as well as bacterial and cell adhesionstudies. The results suggest that the PDMS-QPDMAEMA surface exhibited durablewettability and stability, as well as significant antifouling properties, compared with thenative PDMS and PDMS-PDMAEMA surfaces. In addition, our results present possible usesfor the PDMS-QPDMAEMA surface as adhesion barriers and antifouling or functionalsurfaces in PDMS microfluidics-based biomedical applications.3. A new antifouling polyester monomethoxy-poly(ethylene glycol)-b-poly(l-lactide)-b-poly(sulfobetaine methacrylate)(MPEG-PLA-PSBMA) was obtained by ring-openingpolymerization of L-lactide, and subsequent click chemistry to graft the azideend-functionalized poly(sulfobetaine methacrylate)(polySBMA) moieties onto the alkyneend-functionalized MPEG-PLA (MPEG-PLA-alkyne). The chemical structure of the polymerwas characterized using1H nuclear magnetic resonance (1H NMR) and Fourier-transforminfrared spectroscopy (FT-IR), and its physical properties (including molecular weight, glasstransition temperature, and melting point) were determined using gel permeationchromatography (GPC) and differential scanning calorimetry (DSC). To investigate itshydrophilicity and stability, as well as its antifouling properties, the polymer was alsoprepared as a surface coating on glass substrates. The wettability and stability of this polyester was examined by contact angle measurements. Furthermore, its antifouling properties wereinvestigated via protein adsorption, cell adhesion studies, and bacterial attachment assays. Theresults suggest that the prepared zwitterionic polyester exhibits durable wettability andstability, as well as significant antifouling properties. The new zwitterionic polyesterMPEG-PLA-PSBMA could be developed as a promising antifouling material with extensivebiomedical applications.4. In this study, we prepared four new polyesters: poly(sulfobetaine methacrylate)-,poly(2-methacryloyloxyethyl phosphotidylcholine)-, poly(ethylene glycol)-, and quaternizedpoly[(2-dimethylamino)ethyl methacrylate]-grafted poly(propargyl glycolide)-co-poly(ε-caprolactone). The synthesis was conducted through ring-opening polymerization ofacetylene-functionalized lactones and grafting using click chemistry. The chemical structuresof the polyesters were characterized through nuclear magnetic resonance (1H NMR) andFourier-transform infrared spectroscopy (FT-IR), and their physical properties (includingmolecular weight, glass transition temperature, and melting point) were determined using gelpermeation chromatography (GPC) and differential scanning calorimetry (DSC). For studieson their hydrophilicity, stability, and anti-bioadhesive property, a series of polymeric surfacesof these polyesters was prepared by coating them onto glass substrates. The hydrophilicityand stability of these polyester surfaces were examined by contact angle measurements andattenuated total reflection Fourier-transform infrared spectroscopy (ATR-FT-IR). Theiranti-bioadhesive property was investigated through protein adsorption, as well as cellular andbacterial adhesion assays. The prepared polyesters showed good hydrophilicity andlong-lasting stability, as well as significant anti-fouling property. Thus, the newly preparedpolyesters could be developed as promising anti-fouling materials with extensive biomedicalapplications.In conclusion, in the present study we have successfully synthesized a series of polymerswith different biological function, constructed a series of poymer interface, and explored theirapplication in the life analysis. The study was carried out for different biological functionpolymers preparation methods and techniques, the properties of biological function polymersand its biological applications in-depth study of the system. At the same time, it analysis anddiscuss the further application of these polymers in the biomedical field.更多还原