共轭聚合物-DNA自组装体系的制备、光学性质表征、及药物的控制性释放

Fluorescent nanoparticles based on π-conjugated polymers (CPs) have been developed in recent years. Owing to the highly delocalized π-conjugated backbones, CPs show superiority over small organic dyes in terms of higher absorption, better photostability, larger Stokes shift, and stronger intermolecular interactions. Recently, as one of the most important bio-imaging materials, CP nanoparticles have been demonstrated comparable to inorganic quantum dots in fluorescence quantum yields. However, in the field of stimuli-responsive drug delivery, very little work has been done on CP nanoparticles. Compared with synthetic polymers, DNA exhibits many unique properties, including its biological function, biocompatibility, and molecular recognition capacity. Thus in this project we plan to design and develop a kind of novel nanoparticles based on conjugated polymers and DNAs, which will show multiple functions of selective cancer cell recognition, bio-imaging, and stimuli-responsive drug delivery. First, several conjugated polymers showing near-IR fluorescence or photoacoustic effect will be designed and synthesized, in order to realize deep tissue penetration. Condensed DNA/RNA nanostructures are produced from rolling circle amplifications (RCAs). Next, different supramolecular strategies are developed to fabricate the self-assembling systems of CPs and DNAs; and the most stable and highly fluorescent nanoparticles will be screened for the further modifications. The built-in multi-functional sequences can be readily induced in the DNA/RNA building blocks through designing the templates for RCA reactions. We will introduce the gene drug sequence (for example, antisense oligonucleotides, SiRNAs, or ribozymes), the cancer cell-targeting aptamer sequence for selective cancer cell recognition, and the recognition sequence for endonuclease enzymes, into the DNA/RNA building blocks. As parts of the nanoparticles, the “protected” enzyme nanocapsules will be included, in which the endonuclease is encapsulated into a positively charged thin polymeric shell cross-linked by acid-degradable or reducing-degradable cross-linkers. Once the nanoparticles are delivered to cells, the endonuclease enzyme will be “activated” and cut DNA/RNA building blocks to a high volume of gene drugs.

基于共轭聚合物的荧光纳米粒子是生物成像纳米材料家族中的新成员，由于其生物相容性良好、毒性低、光捕获能力强、光稳定性好等特点，受到了越来越多的关注。目前，这类有机荧光纳米颗粒的最高荧光量子效率已经达到了和无机荧光量子点相同的水平;但相对于无机纳米粒子，有机纳米粒子在药物输送、疾病诊疗方面的开发研究还很欠缺。本课题拟将共轭聚合物引入DNA纳米结构中，开发具有药物可控释放功能的荧光纳米材料。在现代纳米技术中，DNA不仅仅是一种生物大分子，更是一种最智能、最特异、最可控的新型材料。研究表明恒温滚环扩增反应（RCA）制备的高浓度、高分子量的DNA更易形成热力学稳定的纳米结构。因此本课题拟利用RCA方法制备DNA，并发展超分子方法制备共轭聚合物和DNA的自组装体系，获得具有高荧光量子效率和优良稳定性的纳米颗粒；设计特定的DNA序列，赋予荧光纳米材料对癌症细胞特异选择性和可控性的药物释放功能。

项目结合π-共轭功能分子和DNA纳米技术制备新型的有机纳米药物用于癌症治疗与成像。有机纳米药物由于其良好的生物相容性、低毒性、以及多样且优良的性质，受到了广泛的关注。其中π-共轭功能分子在光热及光动力治疗、荧光及光声成像方面有重要应用，通常疏水的π-共轭功能分子需要双亲性嵌段聚合物的包覆实现水相中的稳定分散。近年来生物基大分子，如蛋白、多肽、壳聚糖、多巴胺等被广泛应用于纳米药物载体包覆π-共轭功能分子，除了可以提供相对于人工合成高分子更好的生物相容性外，这类生物大分子还可以提供诸如靶向、肿瘤微环境刺激响应释药等功能。DNA是一种重要的生物大分子，具有结构明确、功能丰富且可控等优点，但还较少被应用于纳米药物载体，我们在开发结合DNA和基于π-共轭功能分子纳米药物方面主要发现包括：（1）经典近红外染料吲哚菁绿在DSPE-PEG-DNA形成的双亲性胶束中被诱导形成完全的J-聚集，其吸收光谱发生了大于100 nm的红移，增加了组织穿透力；同时J-聚集提高了材料的稳定性和光热转换效率，极大增强了光热治疗的效率；最外围的DNA适配体将纳米药物的靶细胞摄取率提高了2倍，增强了肿瘤组织部位的富集能力。（2）利用滚环扩增技术（RCA）制备具有特定功能序列的DNA大分子并用于纳米药物载体。通过RCA模板设计，我们可以在DNA产物中引入癌症靶向的适配体序列，或者对酸性环境刺激响应的序列，制备出具有癌症靶向、肿瘤微环境响应释药功能的纳米粒子；也可以高效地负载siRNA序列，制备出基因沉默效率>80%的纳米药物。这些结果证明利用DNA大分子取代双亲性嵌段聚合物制备多功能性、智能性纳米药物的可行性。最后我们探索了DNA大分子载体和π-共轭功能分子结合的最佳方法，由于π-共轭功能分子极强的疏水性，通过和一段寡聚核苷酸共价接枝可极大改善π-共轭功能分子的水溶性，进而可以和DNA大分子通过序列互补有效复合，该方法可以较好分散π-共轭功能分子的同时保留了DNA的功能性。