仿病毒shRNA递送系统联合基于FRET的光动力治疗肿瘤的研究

The successful tumor gene therapy relies on the development of safe and efficient gene delivery systems. The delivery systems struggle to overcome a multitude of barriers ranging from extracellular barriers (circulation and tumor microenvironment) to intracellular barriers, and achieve efficient transfer of therapeutic genes into target tumor cells. To encounter numerous barriers en route to their target is crucial for access tumor gene therapy, high tumor localization and long wavelength absorption of photosensitizers are also very important for tumor photodynamic therapy. Inspiration by the structure and function of SV40 (non-enveloped virus), we propose a novel approach based on multifunctional artificial virus for combining gene therapy and photodynamic therapy treatments against cancer for the first time in this study. After quantum dots modified shRNA encapsidation by fluorinated /arginine modified G2 lysine dendrons with photosensitizer core, the tumor penetrating iRGD peptide was loaded to construct multifunctional artificial virus shRNA delivery systems. Based on the FRET mechanism (quantum dots as donors and photosensitizers as acceptors), the systems could be applied for photodynamic therapy of tumor, simultaneous shRNA tracing and fluorescence imaging. The study was to further explore the multifunctional artificial virus gene delivery systems, to achieve gene therapy and FRET-based photodynamic therapy. It is important to reveal the relationships between gene therapy and photodynamic therapy. The obtained results could be served as the basis for the further research on the combination of different treatments against tumor.

针对肿瘤治疗中非病毒基因递送系统需克服多重生理屏障，难以实现外源基因在靶点的高效表达，且单一基因靶点不足以治疗肿瘤。而光动力治疗具有广谱性，但存在光敏剂不能有效富集于肿瘤部位且激发波长过短无法有效穿透组织的问题，以及目前单一治疗方案的抗肿瘤效果并不理想的现状。受无包膜病毒SV40蛋白质衣壳的结构和功能的启发，本项目首次利用基于光敏剂为核的功能化（氟烃化修饰或精氨酸修饰）肽类树状大分子和量子点修饰（或负载）的shRNA，并模仿衣壳蛋白装配RNA，以及肿瘤组织靶向渗透肽iRGD，构建多功能仿病毒shRNA递送系统，同时实现基于FRET原理（量子点为供体，光敏剂为受体）的shRNA示踪、荧光成像和肿瘤光动力治疗。该研究在进一步探索仿病毒基因递送系统构建的基础上，巧妙设计基于FRET的光动力治疗，对揭示基因治疗和光动力治疗瓶颈现象有重要意义，同时也为研究和发展多手段联合应用治疗肿瘤提供新的思路。

针对目前单一治疗方案的抗肿瘤效果并不理想的现状：一方面基因治疗中非病毒基因递送系统需克服多重生理屏障，难以实现外源基因在目标靶点的高效表达，且单一基因靶点不足以治疗肿瘤；另一方面光动力治疗虽具有广谱性，但存在光敏剂不能有效富集于肿瘤部位且光敏剂难以有效渗透进入肿瘤组织，以及肿瘤组织处于缺氧状态无法为光动力治疗提供所需的氧气。本项目利用基因治疗与光动力治疗有机结合的方案，发展了肿瘤治疗的崭新策略。本项目圆满完成了研究计划，达到了预期目标，取得了一些创新性成果。在进一步探索仿病毒基因递送系统的设计原理的基础上，构建了能逐级突破基因递送过程中复杂生理屏障的多重生物响应的靶向基因递送系统，研发了活性氧响应的肿瘤缺氧靶向基因递送系统，揭示了多组分之间的相互关系、影响因素与作用规律，以及分子结构与克服基因递送屏障的关系和作用机制，为研发低毒高效的新型仿病毒基因靶向递送系统奠定理论基础。在探索天然糖肽聚合物介导的基因编码的光动力治疗的基础上，还进一步探索了基于天然糖肽聚合物的智能可控富氧纳米平台的设计与构建原理，揭示了光动力治疗中光敏剂不能有效富集于肿瘤部位，深层肿瘤治疗效果不理想及副作用的瓶颈问题及其解决策略；在细胞或亚细胞水平上阐明了光动力学治疗机理，光敏过程引起细胞凋亡机制，基因与光动力联合治疗的分子机制，同时，建立了基因/光敏剂递送系统的构建技术与方法，为建立基因与光动力联合治疗系统奠定一定的理论基础，同时也为研究和发展多手段联合应用治疗肿瘤提供了一定的参考依据。