一种新型靶向肿瘤(干)细胞纳米载体的构建、生物效应评估及光动力治疗的分子机制

High target specificity and efficacy are critical for photodynamic cancer therapeutical system. We have developed a surface porous hollow silica nanosphere (HSNS) drug delivery system for cancer treatment. Loaded with photosensitizer, this system possesses excellent biocompatibility and tumorolytic effect. In order to improve the targeting effect of the system, we have established a triple system by conjugating nucleotide aptamer (Apt) and photosensitizer to the silica nanosphere, which showed highly specific binding and cell penetration efficacy. In this project, flow cytometry combined with exponential enrichment (FCM-SELEX) technology will be utilized to screen and select the aptamer which can be strongly and specifically conjugated to tumor stem cells. The Apt synthesis method for HSNS is critical in this project. And Apt-HSNS compound targeting transportation mechanism after being conjugated with photosensitizer will also be further investigated in cultured cells in vitro, in nude mice, wild type mice and the mice with spontaneous colon tumor in vivo. The biocompatibility, biological security, phototoxicity and biological mechanism of this combined system will be determined by analyzing its chemical composition, morphology, structure and physicochemical characteristics in different biological microenvironments. These data along with its in vivo biodegration, metabolism and excretion will provide solid experimental and theoretical evidence for developing a novel, selective and potent drug delivery system for cancer therapy.

靶向特异和高效能性是肿瘤光动力治疗的决定性因素。我们发明了一种表面多孔的氧化硅纳米空心球(HSNS)药物输运系统，该系统负载光敏剂后具有优良的生物相容性和肿瘤细胞杀伤能力。为增强该系统的靶向性，我们将该纳米球与核酸适配体偶联。体外细胞实验显示此偶联系统具有很高的肿瘤细胞特异性结合和穿透能力。本项目拟进一步采用流式细胞术-指数富集法配基系统进化技术筛选与肿瘤(干)细胞标志物具有高亲和力和强特异性结合的适配体。重点研究HSNS表面修饰肿瘤(干)细胞适配体的合成技术和负载光敏剂后在培养细胞、种瘤裸鼠、野生型小鼠和有免疫力肠道自发性肿瘤小鼠活体内的靶向输运机制。并从组成、形貌、结构和理化性质等方面考察其在不同生物微环境中的生物相容性、安全性、光动力介导毒性作用的生物学机制，以及活体内可降解度、代谢方式和排泄途径，为研制具有肿瘤干细胞特异性识别功能的新型高效安全靶向光敏剂输运系统提供实验和理论依据。

构建特异性靶向肿瘤（干）细胞、性能高效的光敏剂纳米载体是提高光动力治疗肿瘤效果的重要途径。本研究以结肠癌细胞株SW620作为肿瘤干细胞筛选模型，筛选出了CD44+、CD133-结肠癌肿瘤干细胞（CSC），并对其性质进行了分析。用qPCR检测了CSC干性相关基因的表达，完成了结肠癌CSC单细胞克隆成球实验，证明我们获得了结肠癌肿瘤干细胞。采用流式细胞术-SELEX技术来筛选出了两条与肿瘤干细胞特异性结合、有亲和性的适配体。对CSC-Apt-HSNS-PS进行了结构设计与改良，合成了具有壳-核结构，大小为30-100nm球状可均匀稳定分散于超纯水中的纳米复合材料。在此基础上，课题组进一步发明了一种稳定性好、毒性低、热响应强、光成像分辨率高的可用于诊疗一体化的多功能发光纳米载体。将结肠癌干细胞模型和条件性敲入/敲除技术制作的自发性结肠癌动物模型应用于光动力治疗体外和活体评估。研究了负载血卟啉寡聚体（Photosan）、铜半胱胺（Cu-Cy）、原卟啉（PpIX）、卟啉钠（DVDMs）等不同光敏剂对结肠癌及结肠癌干细胞的光动力靶向治疗效果，发现对结直肠癌有明显的杀伤作用，并发现自噬是多种光动力杀伤肿瘤细胞的机理之一。结合高通量RNA测序技术，首次发现了环状RNA（circRNA）参与到光动力治疗中，circHOMER1可作为miR-942竞争性内源性RNA，调节CRC细胞中DLC1的表达，最终调节CRC对PDT的耐药性。我们研制了具有肿瘤干细胞特异性识别功能的新型高效安全靶向光敏剂输运系统，为光动力杀伤肿瘤细胞的临床应用提供理论基础。