【作者】 任科峰；

【导师】 沈家骢； 计剑；

【作者基本信息】 浙江大学 ， 材料学， 2006， 博士

【摘要】 本论文以生物医用装置与基因治疗相结合为背景，采用层层静电自组装技术(Electrostatic layer-by-layer(LbL)self-assembly)和基因技术相结合，构建了负载基因DNA分子的纳米超薄膜，探索了该超薄膜作为原位控释基因传递体系的研究。研究首先采用具有良好生物相容性和生物可降解性的聚赖氨酸(PLL)作为模型聚阳离子和DNA层层组装，构建酶敏感降解的PLL/DNA超薄膜。紫外(UV)光谱和原子力显微镜(AFM)数据显示层层自组装可成功进行，形成具有纳米尺度的超薄膜。通过调节组装溶液中的离子强度，可调控吸附聚电解质的质量，从而可在纳米尺度上调控超薄膜的厚度和DNA的负载量。AFM测试、溴乙啶(EtBr)吸附实验数据显示(PLL/DNA)超薄膜组装过程中，PLL和DNA分子可能相互缠结形成复合物(complex)，该种复合物可有效保护DNA分子，阻止荧光染料内嵌，为安全有效的传递基因物质提供了前提保证。研究进一步探索了PLL/DNA超薄膜的酶降解特性。UV光谱和AFM数据证明，该种超薄膜在蛋白酶溶液中能够逐步降解，超薄膜中的DNA由此得到持续的释放。进一步的研究表明，通过戊二醛交联超薄膜中的PLL，可简单易行的调控超薄膜在酶溶液中的降解速率，从而达到精确调控DNA释放的目的。研究首次提出并证实蛋白酶降解机制控制超薄膜解组装，特有的酶响应机制使DNA的释放具有生物响应性和可控性，使超薄膜在特种酶响应环境下的基因治疗具有巨大的应用前景。聚阳离子型非病毒基因载体所制备的DNA纳米微球由于具有低成本、高安全性等特质，在介导基因转染领域有着广泛的研究。研究将开展通过LbL技术负载聚阳离子为载体的DNA纳米微球，构建(DNA微球/聚阴离子)型超薄膜。首先，实验制备了稳定的，直径在10—30nm左右，表面电位为31.6±6.09mV的聚阳离子聚乙烯亚胺(poly(ethyleneimine)，PEI)为载体的DNA纳米微球。LbL组装过程中，UV光谱和AFM数据表明，DNA微球可以成功吸附到聚谷氨酸(PGA)或聚乙烯苯磺酸钠(PSS)层表面，DNA微球和聚阴离子反复交替的吸附，形成纳米尺度的超薄膜。透射电镜(TEM)显微照片清楚显示了基材表面所构建的超薄膜，平均一个双层的(DNA微球/PGA)和(DNA微球/PSS)超薄膜分别为6和18nm。通过LbL技术温和的组装环境，非常方便的构建出组装有DNA纳米微球的纳米尺度的超薄膜，并且最大程度保持了DNA微球的缔合状态，为该种DNA微球在生物体内的传递提供了强有力的技术平台。研究最后考察了(PLL/DNA)和(DNA微球/聚阴离子)两种类型超薄膜的细胞转染能力。研究首先考察了具有生物酶可降解性的(PLL/DNA)超薄膜。实验结果表明，将超薄膜酶降解，从中释放的DNA分子仍保持有原有的生物活性，可转染细胞。研究接着考察了同样具有生物可降解性的(DNA微球/PGA)超薄膜。实验结果表明，通过蛋白酶的降解，DNA微球可从超薄膜中释放出来，并且该种DNA微球保持了其特有的缔合状态，可直接转染细胞，而无须加入新的帮助转染的载体。进一步的研究表明，(DNA微球/聚阴离子)型超薄膜也可原位转染贴壁于上的细胞。基于PSS的超薄膜表面由于可提供更高密度的DNA微球，因此可以达到比基于PGA的超薄膜更高的原位转染率。通过将(DNA微球/PGA)超薄膜组装在三维聚乳酸组织工程支架上，实现了细胞在组织工程支架表面的转染。对基因超薄膜的细胞转染研究，清楚显示其作为DNA传递体系在基因治疗领域有着切实可行的应用前景。本论文的研究工作成功构建了具有酶敏感特性的(PLL/DNA)超薄膜和高效负载DNA微球的(DNA微球/聚阴离子)超薄膜。两种类型的基因超薄膜可与生物医用装置相结合，在不久的将来，该技术不但有望解决医用装置本身在生物相容性和生物功能化方面所存在的种种不足，同时也将极大的拓展生物医用装置在基因治疗领域中的应用。更多还原

【Abstract】 It is well known that gene therapy is a potential powerful tool to deal with many severe diseases such as cancer. Therefore it has been extensively researched worldwide. Effective gene delivery is the main objective in gene therapy. Toward the goal of constructing novel gene delivery system, two kinds of ultrathin DNA film via electrostatic layer-by-layer (LbL) self-assembly technique have been constructed.Firstly, (DNA/poly-L-(lysine) (PLL)) ultrathin film is constructed via LbL self-assembly technique. The PLL is chosen as model polycation because of its biocompatibility and biodegradability. Therefore, the PLL could endow the film with special ability of enzyme sensitivity. Both UV-vis spectrum and atomic force microscope (AFM) measurements show a typical LbL deposition of DNA and PLL. It is found that the salt concentration of the deposition solution has significant effect on the construction of the films, which might be attributed to the effect of salt ions on the conformation of polyelectrolytes and interaction between PLL and DNA molecules. Therefore, through the controlling salt concentration, the film’s thickness and adsorbed amount of polyelectrolytes could be modulated in nano-scale. The AFM measurement and ethidium bromide (EtBr) adsorption show that the PLL/DNA complexes could be formed during the deposition of film. Such complexes might serve a mechanism to protect DNA from the attack of EtBr. Further experiment of enzyme degradation experiments are carried out to investigate the enzymatically sensitive ability of the film. The data of UV-vis spectrum and AFM show that the (PLL/DNA) film is degraded under enzyme solution and then the DNA is released from the film. After that, it is found that, through the method of glutaraldehyde (GA) cross-linking, the stability of the film could be controlled. More important, it significantly affects the profiles of DNA release.The polycation-based non-viral DNA nanoparticles, which is proved to be effective both in vitro and in vivo, is receiving increasing attention because of several advantages, such as ease of manipulation, low cost, and safety. Second part of our researches, toward the goal of construction of novel delivery system for polycation-based non-viral DNA nanoparticles, (DNA nanoparticles/polyanion) film is constructed via LbL self-assembly technique. Special DNA nanoparticles are prepared with the diameter of 10-30 nm and zeta potential value of 31.6 ± 6.09 mV.Poly(ethyleneimine) (PEI) is chosen as polycation to condense the DNA to nanoparticles. AFM and UV-vis measurements show that the DNA nanoparticles can be successively adsorbed onto the polyanion {poly-L-(glutamic acid) (PGA) or poly(styrene sulfonate) (PSS)} layer to form ultrathin films. The transmittance electron microscope (TEM) measurement clearly shows the cross-sectional films. The average one bilayer of (DNA nanoparticles/PGA) and (DNA nanoparticles/PSS) films is about 6 and 18 nm, respectively. The well-structured, easy processed multilayers with the non-viral DNA nanoparticles may provide a novel approach to precisely control delivery of DNA, which may have great potential for gene therapy applications in tissue engineering, medical implants, etc.Further, the cells culture experiments are carried out to investigate the transfection efficiency of both (PLL/DNA) and (DNA nanoparticles/polyanion) films. Firstly, experiment data confirm that the DNA that is released from the (PLL/DNA) film through enzymatic degradation can transfect the cells very well with the PEI vector, which means that the construction and deconstruction procedure will not affect the transfection ability of DNA. Secondly, through the enzymatic degradation of (DNA nanoparticles/PGA) film, the DNA nanoparticles can be released from the film and remain its complex configuration. Therefore, these released DNA nanoparticles can directly transfect the cells without help of other vector any more. An in situ cell culture experiment suggests that (DNA nanoparticles/polyanion) films have the ability of transfecting cells in situ. Because of the higher DNA nanoparticles density, film based on PSS leads to higher transfection efficiency in situ than that of film based on PGA. Through constructing the (DNA nanoparticles/PGA) film onto the PLA tissue engineering scaffold, the cells transfection on the scaffold can be achieved.In brief, the DNA or DNA nanoparticles contained films could be served as a novel gene delivery system. More important, these kinds of film could be deposited onto almost any kind and any shape of matrix surface, such as tissue engineering scaffold and interventional therapy devices, and therefore be served as a bridge between gene therapy and biomedical devices. The researches might not only figure out the problems induced by biomedical devices itself, but also advance the clinical application of biomedical devices in the field of gene therapy.更多还原