手性γ肽核酸多功能载体的构建及其在微流控芯片中对肿瘤细胞的作用研究

Peptide nucleic acid (PNA) is an antisense drug capable of specifically silencing gene expression. This work aims to solve the problems of PNA in practical application, such as poor tumor cells-targeted drug delivery, difficulty in entering the cells and uncontrollable drug release, by designing and constructing diketopyrrolopyrrole (DPP)-based multifunctional nanocarriers for delivery of PNA. In this work, two mixed DPP monomers, one is modified with diethylene glycol (miniPEG)-grafted chiral γPNA by disulfide linker and the other one with folic acid, can produce an novel nanocarrier in water via copper ions-induced DPP aggregation. Folic acid coat gives nanocarrier the capability of targeting tumor cells, while miniPEG gives nanaocarrier the high water solubility and stealth property, and also avoids the cytotoxicity of conventional polyethylene glycol with large molecular weight. This work studies the interaction between nanocarrier and cells by microfluidic chip capable of making different concentration gradient. In the tumor cells, the cleavage of the disulfide linker nanocarrier will be triggered by high concentration of thiols, most notably glutathione, which can release PNA from the surface of nanocarrier slowly and inhibit the growth of tumor cells. In addition, this work also studies the dissociation process of nanocarrier induced by coordination between copper ions inside the nanocarrier and thiols in tumor cells. The dissociation can recover two-photon imaging of DPP molecules, which is beneficial to the real-time monitoring of interaction between γPNA nanocarrier and cells. The goal of this work is to prepare an easily degradable, multifunctional nanocarrier for γPNA with low toxicity and establish a microfluidic chip-based system for rapid and stable drug evaluation , and finally promote the application of γPNA in the medical field.

肽核酸（PNA）是一种能特异性沉默基因表达的反义药物。本项目围绕PNA药物存在靶向性差、难于进入细胞以及释放不可控等问题，构建基于吡咯并吡咯二酮（DPP）的PNA多功能纳米载体。拟将含二甘醇基（miniPEG）的γPNA 通过二硫键修饰吡啶基DPP，与叶酸修饰的DPP共混，经铜离子诱导聚集成γPNA纳米载体。叶酸外衣赋予载体靶向识别肿瘤细胞功能，miniPEG基团赋予载体高水溶性和"隐形性"，同时避免大分子聚乙二醇的细胞毒性。拟使用微流控浓度梯度生成芯片研究载体与细胞的相互作用。肿瘤细胞内高浓度生物硫还原二硫键，缓慢释放γPNA反义药物，抑制肿瘤细胞的增殖；生物硫与铜离子配位诱导载体的解离及DPP双光子成像功能的恢复，实时跟踪载体与细胞的相互作用。本项目旨在制备一种低毒性、易降解的γPNA多功能纳米载体并构建基于微流控芯片的快速、稳定药物作用评估系统，推动γPNA 药物在医学领域的应用。

基于碱基互补配对的基因芯片技术及反义治疗技术需要设计理化性质优异，生物相容性好，识别性能强的核酸序列和科学的药物递送体系。本项目围绕γPNA的探针设计和多功能药物载体的稳定性、选择性释放药物、载体成像及药物评估等关键科学问题，拓宽γPNA在生物医学领域的应用。首先，合成了基于Fmoc保护的miniPEG基团修饰的手性γPNA单体和序列。其次，通过Fluent流体力学软件设计了药物浓度稀释梯度微流控芯片，为开展载药实验与细胞实验提供了研究基础。基于介孔纳米二氧化硅(MSN)，DPP衍生物和盐酸阿霉素制备了一种新型的刺激响应型的纳米载药系统。载体中的吡啶基吡咯并吡咯二酮衍生物可同时发挥门控及成像作用，实现了纳米载体选择性释放药物和载体示踪。同时，本项目进一步推进了手性γPNA芯片的制备与应用探索，为γPNA药物递送系统的构建提供基础，有望推动γPNA在生物医学中的应用。本项目所获得的研究成果，已发表ESI期刊论文18篇，其中带有项目第一资助标记的17篇，IF≥10的4篇；申请了专利12项（发明专利10项），授权专利6项（发明专利4项）；培养毕业博士研究生3名，毕业硕士研究生5名，在读博士研究生3名，在读硕士研究生5名。