纳米载药系统是一种新型的药物传输系统，可解决难溶性药物的递送问题并实现缓释及靶向性。已进行的纳米毒性研究主要集中在研究无机纳米载体对环境的毒性，而对于小尺寸即粒径小于100纳米，可生物降解的用于载药治疗使用的纳米载体，对其跨膜转运机理及毒性认识有限。本研究选取了香豆素纳米晶体和多肽修饰聚合物胶束作为代表性纳米载体，希望通过对其跨膜转运机制，在亚细胞器中的定位，在生物体内的分布，代谢和消除过程的研究来和其潜在的生物毒性关联。本课题将利用合作双方课题组的优势，结合微观分子细胞模型和宏观体内动态斑马鱼模型，深入阐明构建的两种小尺寸纳米载体在单层极性上皮细胞的入胞，胞内转运及出胞过程；通过细胞器染色和共聚焦显微镜来确定纳米载体在细胞转运过程中的亚细胞水平定位；通过荧光显微镜对斑马鱼进行活体实时观察；通过荧光检测器对斑马鱼体内的纳米荧光粒子进行定量，阐明纳米化颗粒在斑马鱼体内的吸收分布和消除过程。

Nano-carrier based drug delivery system (DDS) has been developed as novel DDS to load poorly-water soluble drugs for sustained and targeted drug delivery in recent years. Although many studies have demonstrated that inorganic and metal nanoparticles have potential toxicity to the environment, few of the studies have been performed to investigate the toxicity and transport mechanism of drug-loaded biodegradable nanoparticles (i.e. <100 nm) for therapeutic application. In the present study, nanocrystals and modified micelles loaded with the high lipophilic fluorescence compound coumarin 6 (C6) were selected as the two delivery systems to explore their transport mechanisms and biodistributions. This project will take advantages of two research groups’ experience and perform mechanistic study at molecular, cellular and in-vivo animal model levels. Specifically, small sized nanocrystals and micelles with particle size less than 100 nm will be fabricated by anti-solvent and film dispersion method, respectively. Their transport mechanism will be investigated on MDCKII and Caco-2 cell monolayers as well as on the zebrafish models by incubation with various inhibitors to elucidate the pathway of endocytosis, transcytosis and exocytosis process. Cellular uptake and transportation of the C6 nanoparticles will be observed by fluorescence/confocal microscope and quantified by fluorescence detector, respectively. Intracellular tracking of C6 nanoparticles will be performed after staining with various probes. The biodistribution of nanoparticle in zebrafish model will be observed by fluorescence microscope and quantified by fluorescence detector to study absorption, distribution and elimination process of the nanoparticles.

随着纳米技术在药物递送领域的高速发展，其展现出相对于传统制剂的独特优势。尽管纳米载体有很多的优点，但目前研究者对纳米载体的跨膜转运机理，转运完整性及毒性认识非常有限。因此本项目旨在研究纳米制剂跨膜转运机制和纳米毒性。我们首先制备了纳米晶体和聚合物胶束两种纳米递药体系，然后研究了纳米制剂对MDCK 细胞和Caco-2 细胞紧密连接的影响，细胞的跨膜转运机理研究，纳米制剂对斑马鱼的毒性研究，以及纳米制剂的体内过程。研究发现，纳米晶体对细胞的存活和细胞膜完整性没有明显的影响。相对于原药或微米级药物颗粒，纳米晶体不仅可以提高其跨膜转运量，也可以改变药物转运的途径，且粒径较小的纳米晶更能促进药物的跨膜转运。纳米晶体在斑马鱼体内过程的研究也显示，小粒径的纳晶具有更好的转运效率，其在成鱼体内的消除过程主要在胆囊中进行。对于PEG-b-PCL胶束在MDCK细胞跨膜转运研究显示，部分胶束以完整的形式跨过细胞单层膜。而配体修饰后的胶束摄取及转运能力与靶头修饰密度相关。配体修饰胶束在斑马鱼体内研究显示，修饰后聚合物胶束可通过与转铁蛋白的相互作用增加其全身摄取量，且在转铁蛋白表达较高的区域（如眼部，肠道和脑部）随着修饰比例增加而增加。而当修饰密度达到一定比例后，这种促进作用将会降低。成鱼肠道跨膜完整性研究显示，胶束以部分完整的形式被吸收进入体内。通过以上的研究工作，我们不仅可以在细胞水平对小粒径纳米制剂的转运机制，毒性研究进行深入的阐述，同时对纳米制剂的评价扩展到在体斑马鱼模型中，建立了不同发育阶段斑马鱼动态可视化模型来研究纳米颗粒的跨膜转运机制，体内分布以及毒性。同时还利用荧光共振能量转移技术研究纳米颗粒在体外及体内模型的完整性。我们相信，斑马鱼模型对将来纳米制剂体内转运以及毒性评价，和载体材料的筛选和开发有重要的指导意义。本项目执行期间，双方共联合发表SCI论文5篇，中文核心期刊2篇。双方合作培养了2名硕士，2名博士，还在国内外学术会议上进行了多次成果交流展示。