【作者】 胡晓芬；

【导师】 沈家骢； 计剑；

【作者基本信息】 浙江大学 ， 高分子化学与物理， 2010， 博士

【摘要】 采用多功能薄膜涂层实现抗凝血、内皮细胞诱导和局部药物控释是解决心血管植入材料临床应用问题的重要手段。本文以分子末端带有大量羟基的超支化聚醚为研究主体,通过对超支化聚醚末端官能基的改性与活化,采用静电和共价键层层组装,制备了多功能薄膜涂层材料,探索其应用于心血管植入体表面涂层的可行性。一.类肝素超支化聚醚静电层层组装构建多功能薄膜涂层材料的研究研究首先对超支化聚（3-乙基-3-羟甲基环氧丁烷）（HBPO）的末端官能团进行磺酸基改性,将疏水的HBPO转化为水溶性的类肝素超支化聚醚HBPO-SO₃。通过荧光探针法、粒径分析和透射电镜（TEM）对聚合物在水溶液中的自组装行为进行表征,证明当HBPO-SO₃的浓度高于临界胶束浓度（CMC）0.017 mg/mL时,可自发组装形成尺寸在25～70 nm之间,内核为疏水的超支化聚醚链,外层为带负电荷的磺酸基的球形胶束。荧光探针法的结果进一步表明,HBPO-SO₃的单分散大分子状态及其自组装聚集体均为包载疏水的客体小分子提供了良好的疏水环境。复钙化凝血时间的测定结果证明了改性超支化聚醚的类肝素性质,分子末端的磺酸基使得HBPO-SO₃体现了良好的抗凝血性;同时体外细胞活性测定和细胞形貌观察证明HBPO-SO₃具有低的细胞毒性和良好的细胞相容性,适用于与血液直接接触的体内环境。超支化聚醚的磺酸化改性不仅改善了聚合物的生物相容性,同时分子末端的大量磺酸基使聚合物具有强聚电解质的性质。以HBPO-SO₃作为肝素的替代物,与聚阳离子壳聚糖静电交替组装制备层状多层膜。原子力显微镜（AFM）数据表明,HBPO-SO₃以自组装聚集体的形式载入多层膜,在多层膜中形成大量纳米尺寸的疏水区域。多层膜可通过“预包载法”和“扩散法”两种方式包载疏水模型药物—芘。荧光光谱和紫外光谱的结果显示,与“预包载法”相比,由“扩散法”制备的芘负载的多层膜具有更高的包载量和更好的控释能力。通过相同的“扩散法”负载方式,在多层膜中包载具有抗再狭窄功能的疏水药物一普罗布考,与肝素/壳聚糖多层膜相比,HBPO-SO₃/壳聚糖多层膜由于具有纳米尺寸的疏水区域,对扩散有机溶剂没有限制,可成功实现对普罗布考的包载和控释。复钙化凝血时间的测定结果表明,即使以抗菌的壳聚糖为最外层,多层膜表面也具有良好的抗凝血性。HBPO-SO₃/壳聚糖多层膜可望成为具有抗菌性、抗凝血性和局部控释疏水药物的能力的多功能涂层材料。二.反应性超支化聚醚共价键层层组装构建多功能薄膜涂层材料的研究研究进一步利用超支化聚醚HBPO的多末端羟基,制备了与氨基化合物具有化学反应活性的端对硝基苯酯基超支化聚醚HBPO-NO₂。在氨基化的基材表面,HBPO-NO₂与PEI通过共价键层层自组装过程,形成了具有共价键交联结构的稳定的多层膜。HBPO-NO₂/PEI多层膜兼容了疏水超支化聚醚与阳离子型聚电解质两种不同属性的物质,因此具有独特的包载性能。多层膜可通过质子化作用包载阴离子型模型药物一丽春红2R,调节载药溶液的pH值可以影响丽春红2R在多层膜中的包载量和释放量,在中性pH值条件下,多层膜即可实现高载药量及可控释放。多层膜可作为原位制备纳米银粒子的模板,纳米银的载入量随多层膜中PEI层数的增加而增加。多层膜还可成功包载疏水药物普罗布考,并实现长效释放。内皮细胞的体外培养结果显示,HBPO-NO₂/PEI多层膜具有阻抗细胞粘附的作用,随着膜厚的增加,这种作用愈加明显。在（HBPO-NO₂/PEI）_n/HBPO-NO₂多层膜表面接枝生物活性分子胶原和多肽REDV,促进了内皮细胞的粘附,改善了多层膜表面的细胞相容性。尤其,胶原功能化的（HBPO-NO₂/PEI）₅/HBPO-NO₂多层膜表现出显著促进内皮细胞粘附的能力。因此,具有化学反应活性的HBPO-NO₂/PEI多层膜不仅可以作为多种治疗因子的局部控释载体,而且可通过表面生物功能化改性调控细胞行为,具有作为心血管植入体表面多功能涂层的潜在应用价值。更多还原

【Abstract】 Much attention has been paid to the application of layer-by-layer（LBL） films as functional surface coatings of implanted medical devices（such as vascular catheters, vascular implants,and heart valves）.They appear as interesting candidates for the localized delivery of therapeutic agents,antibacterial and anticoagulation applications, control of cell adhesion and growth,and so on.Toward the goal of constructing multifunctional LBL films,hyperbranched polyether is used as a building block for LBL assembly,and multilayer films via electrostatic or covalent LBL assembly technique have been constructed,respectively.Construction of multifunctional coatings via the electrostatic LBL assembly of heparin-like hyperbrabched polyetherA water soluble sulfonated hyperbranched polyether（HBPO-SO₃）,consisting of a hydrophobic hyperbranched poly（3-ethyl-3-oxetanemethanol） core and sulfonate terminal groups,is designed as a promising heparin-like biomaterial.The micellization of the resultant HBPO-SO₃ in aqueous solution is monitored by fluorescence spectroscopy using pyrene as a hydrophobic probe and the critical micelle concentration（CMC） is determined to be 0.017 mg/mL.The analyses of transmission electron microscopy（TEM） clearly show that HBPO-SO₃ can aggregate into spherical micelles with diameters of 25～70 nm above CMC.Results from the plasma recalcification time assay and cell culture declare that HBPO-SO₃ exhibits good hemocompatibility and low cytotoxicity.As an alternative to heparin,sulfonated hyperbranched polyether HBPO-SO₃ is employed as a building block to fabricate multilayer films with chitosan via LBL assembly.The atomic force microscopy（AFM） images demonstrate the stability of HBPO-SO₃ micelles during the LBL process and therefore many hydrophobic nanodomains are incorporated into the LBL films to provide nanoreservoirs for hydrophobic guest molecules.The fluorescence emission spectra verify that the hydrophobic dye pyrene can be incorporated into the LBL films either by pre-encapsulation in HBPO-SO₃ micelles or post-diffusion in preassembled multilayer films.Compared with the pre-encapsulation approach,the post-diffusion process is more efficient to incorporate hydrophobic guest molecules into the LBL films and can carry out a much more controlled release of the guest molecules.Hydrophobic drug probucal,which has powerful antioxidant properties and can prevent restenosis after coronary angioplasty,is chosen and incorporated into multilayer films via post-diffusion.Successful loading and controlled release of probucal in HBPO-SO₃/chitosan multilayer films are obtained.In addition,HBPO-SO₃/chitosan multilayer films exhibit anticoagulant activity even with chitosan as the outmost layer. Therefore,a multifunctional coating with potential anticoagulation,antibacterial and localized release of hydrophobic drug is developed.Construction of multifunctional coating via the covalent LBL assembly of reactive hyperbranched polyetherThe construction of multilayer films via the alternating covalent LBL assembly of p-nitrophenyloxycarbonyl groups-terminated hyperbranched polyether （HBPO-NO₂） and polyethylenimine（PEI） onto aminolyzed substrates is investigated. Ellipsometry measurements confirm the successful LbL assembly of HBPO-NO₂ and PEI.Results from contact angle measurements and UV-vis spectrum demonstrate that 5-bilayered multilayer films form uniform surface coatings on the substrate surface. HBPO-NO₂/PEI multilayer films can be used as localized delivery carriers for multiple therapeutic agents because of the distinct properties of the building blocks. The loading behaviors of HBPO-NO₂/PEI multilayer films toward hydrophilic anionic dye ponceau 2R can be controlled by the protonation of PEI,and ponceau 2R-loaded multilayer films prepared at neutral pH value exhibit well loading and release behaviors.By making use of the effective chelating capability of PEI to Ag⁺,Ag⁺ is loaded into the LBL films and then is in situ photoreduced into Ag nanoparticles in the multilayers template.The amount of Ag nanoparticles increases with the increasing of the number of PEI layers in the LBL films.The hydrophobic hyperbranched polyether chains can be regarded as reservoirs for hydrophobic guest molecules,and the LBL films are capable of controlled loading and release of hydrophobic drug probucal.Results from cell experiments indicate that increasing the thickness of the LBL films,human umbilical vein endothelial cells（HUVECs） adhesion and proliferation on the LBL surface are obviously decreased.The multilayer films with HBPO-NO₂ as the outmost layer react readily with the nucleophilic N-terminus of the extracellular matrix proteins collagen or peptide REDV to achieve biomimetic surfaces.The adhesion and growth of HUVECs for the biomolecules-grafted surfaces are better than that of ungrafted multilayer films. Especially,the immobilization of collagen on the surface of 5.5-bilayered multilayer films significantly improves the HUVECs adhesion and proliferation.Therefore,the LBL films exhibit excellent capabilities of localized delivery of multiple therapeutic agents and control of cell function.The facile method here to prepare multifunctional LBL films may have good potential for surface modification of cardiovascular devices.更多还原