基于核酸四面体的肿瘤成像与治疗一体化纳米凝胶系统

Visualized cancer therapy is expected to achieve real-time monitoring of individual treatment, which is of great significance to enhance the cure rate of cancer. How to design and fabricate nano-assemblies to effectively control the effect of cancer diagnosis and treatment in vivo is an important scientific problem to be solved. Based on previous work, this project aimed to construct a tumor microenvironment responsive DNA nanogel system with tetrahedral DNA as the structural unit and intermolecular i-motif structure as the pH response element. The tetrahedral DNA is modified with near-infrared fluorescent molecules and carries multiple miRNA analogues. This DNA nanogel system simultaneously realizes tumor imaging with high specificity and high sensitivity as well as multiple miRNA delivery to tumor cells. By using framework nucleic acids as the nanogel skeleton, it is expected to overcome the shortcomings of traditional DNA hydrogels, such as high gelation concentration and low mechanical stability. Because the transition midpoint and transition range of i-motif conformation are tunable, a well-designed i-motif structure is expected to be responsible to the tiny pH difference between tumor microenvironment and normal tissues. FRET is employed as the fluorescence excitation method in order to enhance signal-to-noise ratio, reduce false positive signals and prolong fluorescence excitation time. The abundant modifiable sites of frame nucleic acids offer the possibility of carrying multiple microRNAs. The successful implementation of this project will realize real-time monitoring of gene therapy and provide an effective scheme for the development of visualized cancer therapy.

癌症可视化治疗有望实现实时监测的个体化治疗，对提高癌症治愈率具有重大意义。如何设计与制备纳米组装体，以实现在活体环境下对肿瘤诊疗效果的有效调控是目前需解决的重大科学问题。本项目拟在前期工作基础上，构建以核酸四面体为结构单元、以分子间i-motif结构为pH响应元件的肿瘤弱酸性微环境响应型DNA纳米凝胶，并且在核酸四面体上修饰近红外荧光分子对、搭载多个miRNA类似物，同时实现肿瘤的高灵敏性成像和对肿瘤细胞的多miRNA递送。本项目的特点在于，以框架核酸结构作为凝胶骨架，有望克服传统DNA凝胶的高成胶浓度和低机械稳定性缺点；基于i-motif的pH响应元件有望对病变与正常组织的微小pH差异作出高灵敏的响应；以荧光共振能量转移作为荧光激发方式，能够提高信噪比，减少假阳性信号；框架核酸结构丰富的可修饰位点为搭载多种miRNA提供了可能。本项目的成功实施将实现实时监测的基因治疗。

如何设计与制备纳米生物传感系统，以实现根据实际应用需求对传感性能的精确定制是目前需解决的重大科学问题。生物传感器中多价受配体结合的协同性对其响应灵敏性有决定作用。双分子的多价受体是人工功能性分子系统的常用工具，但目前协同性的研究中大家几乎都将单分子受体作为模型。另一方面，监测细胞微小pH变化对于疾病监测和诊断具有重要意义。因此本研究（Chem. Commun., 2021, 57, 3247）以双分子C四链体作为模型分子，考察了其氢离子结合协同性的大小以及可能的调控因素。研究结果有助于制备具有精确设计的性能的分子器件的制备。本研究构建了构建了以DNA四面体为结构单元、以分子间C四链体结构为pH响应元件的高响应型pH传感系统，在细胞层面实现了对该传感系统的检测范围的精确调控。.很多病理过程（如肿瘤生成）伴随着细胞环境pH的变化，检测细胞环境pH变化对于理解和干预这些病理过程具有重要意义。分子传感器的响应曲线取决于两个参数：响应中点和响应区间的大小。而这两个参数往往是固定的，不可调节，这阻碍了在具有生理意义的pH区间（4.5-8.0）中任意范围内pH的检测。针对这一问题，项目负责人将i-motif结构搭载在四面体框架核酸上构建pH传感器，通过改变i-motif的序列可以调控传感器的响应中点，通过混合两种传感器可以调控响应区间的大小，并且通过胆固醇-细胞膜疏水作用将四面体锚定在细胞膜上，该调控机制可用于细胞膜上pH变化的原位监测中（Anal. Chem. 2021, 93, 12170）。.另外，项目负责人对DNA纳米结构的调控方式、生物传感器制备和药物递送三个方面进行了综述（Nano Res. 2022, 15, 3504；NPG Asia Mater. 2021, 13: 42； Chemnanomat, 2022, DOI: 10.1002/cnma.202200459）。

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