基于核酸适配体精准识别的肿瘤靶向生物治疗与化疗协同作用研究

Precise recognition by aptamer to the cell surface target is critical to cellular signaling induction and drug delivery into cells. However, conventional technologies cannot fulfil the challenge and greatly limited the synergistic treatment of tumor-targeted biotherapy and chemotherapy. Our previous studies have suggested that DNA ORIGAMI nanotechnology may be a promising way to manipulate the needed aptamer. Therefore, DNA nanotherapy system will be modified with the aptamer at well-defined positions and number by accurate nanoscale distance manipulation for precise biorecognition (Apt-Drug/DNANTS) to target of the cell surface and such system will be assembled for the synergistic treatment of cancer by biotherapy and chemotherapy. Apt-Drug/DNANTS will be characterized for structure and morphology, encapsulation efficiency, loading capacity and stability. The precise recognition, targeting, affinity and cellular signaling induction of Apt-Drug/DNANTS to cancer cells will be investigated by varying the number and positions of aptamer on the surface of the DNA nanotherapy system. The biodistribution and tumor targeting of Apt-Drug/DNANTS in tumor-bearing nude mice will be assessed by using in vivo live fluorescence image technology. The early immune responses will be explored by determining the level of cytokines such as IFN-α, IL-6 and IL-12 in plasma. The synergistic treatment of cancer with biotherapy and chemotherapy of Apt-Drug/DNANTS will be comprehensively evaluated by in vitro cancer cell viability, in vivo inhibition of tumor growth and survival time of tumor-bearing nude mice. Our investigation will not only clarify the relationship between the number and spatial locations of aptamer and its targeting and biological effects, but also provide a new research thought and treatment strategy for synergistic treatment of the tumor-targeted biotherapy and chemotherapy based on aptamer precise recognition.

核酸适配体(Apt)精准识别靶标是其有效诱导细胞信号和靶向递送药物的关键，然而传统技术无法实现，因此严重影响了其生物治疗化疗协同作用。本课题组前期研究表明DNA纳米技术有望解决这一难题。本课题拟采用DNA 纳米技术使Apt精确修饰载药纳米载体，在纳米尺度范围控制Apt的空间位置和数量，构建可精准识别靶标、具有生物治疗化疗协同抗肿瘤作用的纳米治疗系统。考察影响其结构形态、包载药量、稳定性的因素；研究Apt空间位置和数量对靶标的精准识别、靶向性、亲和力和细胞信号诱导的影响；以荷瘤裸鼠为模型，采用活体成像实时考察其体内靶向性；测定血浆中细胞因子评价其早期免疫反应；体外以肿瘤细胞生存率为指标、体内以肿瘤生长抑制和生存时间为指标，综合评价其生物治疗化疗协同治疗肿瘤的最佳条件。本研究可阐明Apt空间位置、数量与其靶向性和生物效应的关系，还能为Apt精准识别靶向肿瘤的生物治疗化疗协同作用研究提供新思路。

适配体精准识别靶标是其有效诱导细胞信号和靶向递送药物的关键，本项目成功运用DNA纳米技术在纳米尺度使适配体定量定位精确修饰纳米载体构建靶向DNA纳米治疗系统。首先设计研究了不同数量和位置适配体C2NP（Apt）修饰的载阿霉素的可靶向淋巴瘤细胞的DNA纳米管治疗系统，将阿霉素载入到DNA纳米管中后提高了阿霉素的稳定性；载药螺旋DNA纳米管和载药直形DNA纳米管的体外释放研究表明螺旋DNA纳米管对阿霉素有一定的缓释作用，而直形DNA纳米管对阿霉素无缓释作用；DNA纳米管上修饰30个C2NP且相邻之间的距离为6nm的治疗系统（DOX@T-DNT-30Apt-6）能显著诱导K299淋巴瘤细胞p53蛋白表达增加，提高细胞内活性氧水平，阻滞细胞周期中的G2/M期，增强细胞的晚期凋亡率和抑制细胞增殖，同时发挥DOX的化疗和C2NP的生物治疗作用，达到协同增强抗肿瘤效果。另外，本研究又构建了肽适配体（FAS和TFM）定量定位修饰的双靶点DNA纳米治疗系统，其中TD-3(TFM)3(FAS)的FAS以三聚化的形式增强激活Fas/FasL系统从而提高HT-29肿瘤细胞凋亡，同时亦能增强激活p53-Bax/Bcl-2通路而提高AIF/EndoG依赖性凋亡和自噬性细胞死亡，体内外显示出较强的肿瘤靶向性和抗肿瘤效果。上述研究结果表明适配体定量定位修饰构建的DNA纳米治疗系统能有效识别靶标、发挥多机制协同治疗作用，在抗肿瘤治疗中显示了很好的应用潜力。通过本项目研究已发表SCI收录论文5篇，会议论文摘要4篇，最高影响因子为7.901，获得授权发明专利2项，培养研究生5名。以本研究为基础，2018年申报的国家自然科学基金国际（地区）合作与交流项目（81861138004，靶向治疗类风湿性关节炎的核酸纳米抗体研究）获得资助。