金纳米结构/特异性配体自组装病毒样聚集体的构筑、表面等离子共振光谱及其肿瘤靶向、光热疗法原理研究

Great issues remain in current tumor-targeting therapy such as low efficiency, difficulty in tending to the target malignant cell lines and then to kill it thoroughly. This project is to develop a novel approach to solve above problems. Taken colon cancer SW620 as an example, phage monoclones on which display octopeptides on the primary coat protein pVIII with good affinity and specificity are isolated by biopanning the landscape phage library with the target cells. Then, the pVIII fused with specific ligands will be obtained by disassembly phage monoclones and purification by gel filtration. Virus-like nanoparticles (VLPs) with good biocompatibility, low toxicity and stability will be constructed by successive assembly of polyelectrolyte and pVIII or selective ligand-fused pVIII onto gold nanostructures. The surface plasma resonance (SPR) spectra of the self-assembled VLPs will be studied by changing the type of assemble unit and surface layers in order to tune the SPR maxium peak wavelength (λmax) to about 808 nm, the central wavelength of near infrared laser. The relationship between λmax and the type of assemble units will also be investigated. Further, the spatial orientation of the outermost layer of the specific-ligand-fused pVIII will be determined. This project aims at establishing the method of actively targeting the colon tumor SW620 and followed by light-thermal killing it by facilitating VLPs which combine the selective targeting property of fusion pVIII and heating property of gold shell upon absorbing the near infrared light. This study will not only develop a novel tumor-targeting therapy with high efficiency and low toxicity, but also provide a novel idea for constructing a variety of capsid-protein-based functional self-assembled VLP complexes. Meanwhile, the prepared VLPs hold great potential to be used as ideal contrast agents, which may open up new strategies for bio-imaging.

针对目前肿瘤靶向疗法效率低、难以趋向肿瘤彻底杀灭肿瘤细胞的难题，本项目以结肠恶性肿瘤细胞为研究对象，拟提出通过生物淘选获得在丝状噬菌体主要衣壳蛋白pVIII表面展示肿瘤细胞靶向特异性配体的噬菌体单克隆，经纯化获得融合特异性配体的pVIII。在金纳米结构表面组装重组的pVIII，构筑生物相容性好、毒性低、稳定的病毒样自组装聚集体，调控聚集体的等离子共振峰峰位至近红外激光器的中心波长808nm附近，揭示自组装诱导的聚集体的共振峰位与组装基元的种类和自组装层的关系，阐述自组装体最外层pVIII的空间取向，旨在利用pVIII配体的特异靶标识别实现对肿瘤细胞的专一靶向传递和金壳结构的吸收近红外光发热杀灭肿瘤细胞。本项目将创新靶向能力更强, 高效、低毒的肿瘤靶向光热疗法, 为构建其他病毒衣壳蛋白的功能组装体系提供了新思路。本项目开发的自组装病毒样颗粒经荧光染色后可作为造影剂，将为生物成像开拓新途径。

恶性肿瘤的早期诊断和治疗成为临床医学关注的焦点。开发特异性靶向试剂、高灵敏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的重要途径。特异性多肽具有稳定性高、选择性高、易合成等优点，有望成为新型靶向试剂。噬菌体展示技术将外源随机多肽展示于衣壳蛋白表面，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。金纳米结构具有表面等离子共振（SPR）性质，可高效地吸收近红外光(650-900 nm)并转化为热能，可作为制备多功能纳米材料的基础。本项目针对纳米材料在肿瘤早期诊疗中靶向效率低的缺点，以结肠肿瘤细胞SW620为模型，从f8/8风景噬菌体文库中成功鉴定出与靶标细胞SW620特异性结合的八肽序列DDAGNRQP，分离纯化获得该特异性pVIII融合蛋白，其羧基端带正电荷，氨基端带负电荷，整个蛋白带微弱负电荷。利用一步法合成了聚二烯丙基二甲基氯化铵稳定的金银异质纳米棒，其SPR光谱的纵轴共振峰位于803 nm。利用层层自组装技术，在金银异质纳米棒表面依次组装聚苯乙烯磺酸钠、荧光分子罗丹明6G和pVIII融合蛋白。在组装过程中，透射电镜图片显示金银异质纳米棒的最外层逐渐加厚，SPR光谱的纵轴共振峰不断红移，最终被调制至810 nm。所制备的仿生纳米组装体最外层的融合蛋白的靶向八肽N端朝向纳米复合材料的表面。该组装体具有很好的生物相容性，它与SW620细胞孵育后，经4 W/cm2的808 nm激光照射照射10 分钟可有效杀死SW620细胞，低于传统的金纳米棒或纳米银颗粒所需功率，没有吸收仿生纳米组装体颗粒的细胞则依然存活。因此，通过可控自组装，构建了具有靶向荧光成像、专一的肿瘤细胞识别和高效光热杀伤效率的多功能仿生纳米聚集体。