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题名	特异性噬菌体衣壳融合蛋白纳米复合材料的组装及其生物成像和肿瘤光热疗法应用
作者	王菲
学位类别	博士
答辩日期	2015-11
授予单位	中国科学院研究生院
授予地点	北京
导师	刘爱骅
关键词	特异性噬菌体衣壳融合蛋白，SPR光谱，金纳米复合材料，组装，光热治疗
学位专业	生物化学与分子生物学
中文摘要	纳米技术极大地推动生物医学的发展。随着癌症死亡人数的增多，恶性肿瘤的早期诊断和治疗成为临床医学关注的焦点。开发高特异性靶向试剂、高灵敏性成像探针和低毒高效的治疗方法是提高肿瘤检测灵敏度和准确性，有效遏制肿瘤增殖和扩散的重要途径。特异性多肽具有稳定性高、分子量小、选择性高、易合成等优点，有望作为昂贵抗体的替代物，成为新型靶向试剂。噬菌体展示技术能够将外源随机多肽展示于噬菌体衣壳蛋白表面，构建而成的噬菌体展示文库可用于高通量筛选靶标特异性多肽配体。 金纳米结构具有独特的表面等离子共振（SPR）性质，能够高效地吸收近红外光 (650-900 nm) 并转化为热能，同时它具有良好的生物相容性，是目前研究最为广泛的光热制剂，可作为制备多功能纳米材料的基础。因此，本论文针对肿瘤早期诊疗对纳米材料的需求，以结肠肿瘤细胞SW620为模型，制备了噬菌体衣壳蛋白修饰的多功能纳米复合材料，实现对结肠肿瘤细胞的靶向多模式成像和高效光热治疗。 首先，将SW620细胞与f8/8风景噬菌体文库进行生物淘选，挑取多个能够与细胞结合的噬菌体单克隆。经噬菌体捕获测试成功筛选鉴定出SW620细胞特异性八肽配体DDAGNRQP。大量扩增该噬菌体并分离纯化获得展示DDAGNRQP八肽的融合蛋白pVIII（fusion pVIII），其羧基端含四个赖氨酸残基因而带有正电荷，氨基端展示两个天冬氨酸和一个精氨酸因而带有负电荷。与传统抗体相比，该fusion pVIII蛋白不仅具有独特的表面电荷分布，还具有分子量小、稳定性高、特异性、亲和性强、廉价等优点，可作为SW620细胞识别的特异性配体。 其次，由于人体对近红外光吸收最少，因此利用一步法合成了纵轴SPR吸收峰位于803 nm的金银异质纳米棒，表面包裹阳离子聚合物而带有正电荷，以阴离子聚合物为介质，通过层层自组装方式在金银异质纳米棒表面负载荧光分子和定向组装SW620细胞特异性fusion pVIII蛋白。随着组装层的增加，纳米材料的吸收峰逐渐红移，最终所得仿病毒纳米复合材料的SPR长轴吸收峰位于810 nm，因此可应用于体内肿瘤治疗。纳米材料表面电位的变化表明，fusion pVIII蛋白独特的表面电荷分布使其融合有DDAGNRQP八肽的氨基端展示于纳米材料表面。细胞激光共聚焦荧光成像进一步证实，组装形成的仿病毒纳米复合材料在表面特异性八肽的介导下，靶向结合并进入SW620细胞，成功实现对肿瘤细胞的靶向可控荧光成像。细胞光热杀伤检测显示，相比于传统的金纳米棒和银纳米片等光热制剂，仿病毒纳米复合材料具有较高的光热转化效率，能够在功率为4 W/cm2的近红外激光照射10 分钟条件下，利用产生的过高热特异性杀伤SW620细胞。 第三，磁共振成像（MRI）具有分辨率高和无电离辐射的优点，成为临床肿瘤检测的重要方法，因此，开发以MRI为基础的多模式造影剂和光热制剂具有重要意义。利用溶剂热法制备了超顺磁Fe3O4纳米微球，其形貌与表面活性剂和反应物前体密切相关。透射电子显微镜（TEM）和X射线衍射（XRD）结果表明Fe3O4纳米微球是由单个小尺寸Fe3O4纳米颗粒聚集而成，表面活性剂能够减小单个Fe3O4纳米颗粒的尺寸，同时反应物前体中结晶水的存在会阻碍Fe3O4纳米颗粒的聚集。在Fe3O4纳米微球表面修饰SiO2和罗丹明B （RhB）分子，得到具有荧光特性的Fe3O4@RhB/SiO2纳米颗粒。利用蔗糖还原法合成金纳米球，将其通过金硫键稳定组装于Fe3O4@RhB/SiO2纳米颗粒表面并生长。随着金壳的形成和厚度的增加，纳米材料的SPR吸收峰逐渐红移，通过调控金壳生长过程得到吸收峰位于747 nm的Fe3O4@RhB/SiO2@Au纳米复合材料。探讨了复合纳米材料制备过程中的各种影响因素并确定最优制备体系。活化SW620细胞特异性fusion pVIII蛋白的羧基端并定向组装于Fe3O4@RhB/SiO2@Au纳米颗粒表面，其融合DDAGNRQP八肽的氨基端展示于纳米材料表面，所得Fe3O4@RhB/SiO2@Au@fusion-pVIII纳米复合材料的SPR吸收峰红移至774 nm，因此其可用于体内肿瘤靶向成像和治疗。纳米复合材料不仅具有高达176.2 mM-1s-1的弛豫效率（r2），而且能够特异性结合并进入SW620细胞，实现对肿瘤细胞的靶向荧光成像，因此，可作为MRI和荧光成像的双模式造影剂。光热杀伤实验结果表明，Fe3O4@RhB/SiO2@Au@fusion-pVIII纳米复合材料具有较高的光热转化效率，能够在功率为3 W/cm2的近红外激光照射10 分钟条件下，实现对肿瘤细胞的高效靶向光热杀伤。
英文摘要	The emerging nanoscience and nanotechnology hold great promise in advancing biomedicine. As the increasing mortality, the early diagnosis and therapy of malignant tumor has drawn great attention in current cancer treatment. In order to improve the sensitivity and accuracy in cancer diagnosis, and efficiently inhibit the tumor proliferation and migration, it is in urgent need to explore tumor-targeting ligands with high specificity, imaging contrast agents with high sensitivity and resolution, and efficient therapy method with minimum side effects. As an attractive alternative to antibodies, specific peptides show higher stability and selectivity, smaller molecular weight and easier to scale up synthesis. The random foreign peptides can be fused to phage coat proteins by genetic engineering and the constructed phage display library can be used to select specific peptides in a high-throughput way. Gold nanostructures with near-infrared (NIR) absorption (650-900 nm) have been widely used in tumor photothermal ablation owing to their unique SPR property that can convert the absorbed light energy into hyperthermia. Besides, gold nanostructures have been proved to be biocompatible, which promotes their clinical applications. In this project, using the colorectal carcinoma cell SW620 as a model, the specific phage coat proteins-conjugated gold nanocomposites were assembled for cancer cell-specific multimodal imaging and efficiently photothermal therapy. Firstly, the SW620 cells were biopanned with f8/8 landscape phage library, from which several landscape phage monoclones were isolated. According to the results of phage capture test, the SW620 cell-specific phage displaying octapeptide DDAGNRQP was successfully identified. The specific phage was amplified and its pVIII fusion coat proteins (fusion pVIII) were isolated in large quantities. The C terminus of fusion pVIII protein is positively charged because of the existing four lysines, and the N terminus is negatively charged because of the displayed two aspartic acids. Comparing with the traditional antibodies, the specific fusion pVIII proteins not only exhibit the exact charge distribution, but also have advantages of low molecular weight, high stability, high specificity and affinity, and low cost, so they are able to be used as SW620 cell-targeting ligands. Secondly, as the hemoglobin and water in vivo have lowest absorption in NIR region, the Au@Ag heterogenous NRs with longitudinal SPR absorption peak at 803 nm were synthesized by controllable one-pot method, using the cationic polyelectrolyte as the surface stabilizer. The fluorescent molecules and SW620 cell-specific pVIII proteins were attached on the Au@Ag heterogeneous NRs through a layer-by-layer (LbL) assembly. In the self-assembly process, the absorption peak of the nanostructure gradually shifted to longer wavelength, and the ultimately assembled bio-mimetic nanostructures showed maximum absorption peak at 810 nm, which made them ideal agents for in vivo tumor therapy. Due to the unique dipole properties of fusion pVIII proteins, their N terminuses with negative charge that were fused with DDAGNRQP were exposed outwardly on the bio-mimetic nanostructures, which was characterized by the change of zeta potential. The fluorescent result further confirmed that the assembled fusion pVIII proteins can specifically direct the constructed nanostructures into SW620 cells, and successfully used for controllably optical imaging of cancer cells. The cell viability test showed that the bio-mimetic nanostructures exhibited higher photon-thermal conversion efficiency than gold nanorods and silver nanotriangle, and were able to specifically ablate SW620 cells after 10 min illumination with a NIR laser in the light intensity of 4 W/cm2. Thirdly, MRI has become the most important imaging mode of tumor because of the advantages of high spatial resolution and nonionizing radiation. Therefore, it is in great need to explore multifunctional nanocomposites for multimodal imaging and photothermal therapy of tumor. The superparamagnetic Fe3O4 nanospheres were prepared through solvothermal method and the morphology was intensely related to the surfactant and precursor. TEM and XRD datas indicated that Fe3O4 nanospheres were aggregated from many Fe3O4 nanocrystals of small size that can be decreased by adding surfactant. The crystalline water in the precursor inhibited the aggregation of single Fe3O4 nanocrystals. By modification with tetraethoxysilane and RhB, Fe3O4@RhB/SiO2 nanoparticles were formed and their fluorescent intensity was tested. Then, gold nanospheres were synthesized using surcose as reductant and stably assembled onto the surface of Fe3O4@RhB/SiO2 nanoparticles via the Au-S chemical bond to improve the stability and reproducibility of Au shells. As the absorbed gold nanospheres grow in the reduction process, Au nanoshells were formed and their absorption peak shifted to the longer wavelength gradually. By controllable synthesis, Fe3O4@RhB/SiO2@Au nanocomposites with the maximum absorption at 747 nm were successfully prepared. The C terminuses of fusion pVIII proteins were activated and assembled onto the surface of Fe3O4@RhB/SiO2@Au nanocomposites, leaving their N terminuses fused with DDAGNRQP outwardly. The obtained Fe3O4@RhB/SiO2@Au@fusion-pVIII nanocomposites showed SPR peak at 774 nm, which made them suitable for in vivo applications. The T2-weighted imaging showed that the r2 value of the nanocomposite was as hihg as 176.2 mM-1s-1. Moreover, the fluorescent images confirmed that the nanocomposites were specifically endocytosed by SW620 cells. Therefore, Fe3O4@RhB/SiO2@Au@fusion-pVIII nanocomposites can be simultaneously used as constrast agents for both MRI and fluorescent imaging. The cell viability showed that the prepared nanocomposites were able to specifically ablate SW620 cells after 10 min illumination with the light intensity of 3 W/cm2. Therefore, the as-prepared Fe3O4@RhB/SiO2@Au@fusion-pVIII nanocomposites were successfully applied to specifically bimodal imaging and efficiently photothermal therapy of cancer cells.
语种	中文
学科主题	理学
公开日期	2020-12-01
内容类型	学位论文
源URL	[http://ir.qibebt.ac.cn/handle/337004/8081]
专题	青岛生物能源与过程研究所_生物传感技术团队
作者单位	中国科学院青岛生物能源与过程研究所
推荐引用方式 GB/T 7714	王菲. 特异性噬菌体衣壳融合蛋白纳米复合材料的组装及其生物成像和肿瘤光热疗法应用[D]. 北京. 中国科学院研究生院. 2015.

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