基于难溶性药物纳米晶体miRNA胞浆直接递送体系的作用与机理研究

Toxicity, low transfection and poor tissue penetration have become the bottle neck for the clinic use of conventional carriers for miRNA delivery. Here, to improve miRNA delivery fundamentally, we will develop a new formulation with globular-protein coated nanocrystals of poorly water-soluble drugs as carriers. Its potential advantages may be included: (1) directly delivering the system into cytoplasma without entrapment in endosome-lysosome system, (2) small-molecule drug particles with activities can be completely dissolved in body without toxicity, therefore having potential to break through the bottle neck of gene delivery, (3) obtaining combined therapy of small-molecule and gene drugs, and (4) the formulation preparation is easy and reproduced, thus possessing great potential in clinic application. The system will be optimized and characterized by current pharmaceutical technology; moreover, with the help of fluorescence probe quenching in aqueous environments and live-cell CLSM imaging, the cellular entry, cytosol location, and disintegration of carriers will be explored. Via coarse-grained molecular dynamics and patch clamp-AFM technologies, the membrane penetration and disruption will be evaluated. In addition, the compatibility will be studied in the global mapping of protein expression by proteomics technology. The work will provide a new strategy for the gene delivery, which is of great scientific significance for the gene delivery. The structure and design ideas of the system are original since it has not been reported at home and abroad. We not only have strong research-force, but also possess perfect research-conditions for the project. Moreover, the base of relevant work for the project is solid and the primary results indicate its feasibility.

传统的miRNA递送载体毒性明显、转染率低与病灶透过性差。本课题拟采用乳球蛋白包裹的难溶性药物纳米结晶为载体以实现miRNA的高效递送。潜在优势有：（1）绕开溶酶体，将miRNA直接递送至胞浆，实现高效转染；（2）具有活性的小分子纳米药物为载体，安全性无毒，有望突破基因给药的瓶颈；（3）实现小分子药物与基因药物的协同治疗；（4）体系构建简单、易于重现，临床应用前景广阔。运用现代制剂学手段对其进行优化和表征；结合水环境淬灭探针与活细胞成像术等探讨体系入胞、胞浆定位以及解体过程；利用粗粒分子动力学技术与膜片钳-原子力显微镜成像术研究体系的膜渗透性与细胞膜表面损伤情况；采用蛋白组学和病理学等手段评价体系的安全性。该体系将为miRNA的有效递送提供一种全新的策略，具有重大科学意义。本设计思路国内外未见报道。项目新颖性好，研究力量与科研条件具备，工作基础较扎实，已有工作结果表明本项目的可行性良好。

基因药物和蛋白类药物在治疗肿瘤方面发挥重要的作用，然而，现有的基因或蛋白的递送载体在细胞摄取过程中会进入内涵体-溶酶体系统，导致仅有2%的药物最终被释放到细胞质中，这已成为大分子药物递送的主要瓶颈。因此本研究主要提出“药物递送药物”（DDD）系统的概念，以阳离子化的β-LG作为稳定剂，制备PTX棒状纳米晶（PNPs）作为基因（let-7a）和蛋白的递送载体，并将其应用于基因药物和蛋白药物的递送中，DDD递送系统能通过非内涵体-溶酶体的小窝蛋白/脂筏摄取途径进入肿瘤细胞，避免了溶酶体酶对大分子药物的降解，将小分子药物与基因和蛋白的直接递送至细胞质，具有生物相容性好，穿透性强，肿瘤部位蓄积程度高，药效显著等优势，实现了对基因和蛋白安全有效地递送。本课题相关研究内容已发表SCI论文10篇，申请中国专利3项。项目研究内容培养博士研究生2人、硕士研究生4人。

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