基于肠道PEPT1特异性黏附的阿霉素口服转运体纳米粒的构建及转运机制研究

Despite the extensive research and success stories with other routes for drug delivery, the oral route is still the most preferred route because of its basic functionality and the advantages that ensue. PEPT1, a kind of intestinal transporter, has specific affinity with L-amine acids. It's worth noting that PEPT1 has not been used to enhance the oral absorption of nanoparticles by speci?c interactions with the mucosal surface. Moreover, it is not clear that the transport mecanism based on speci?c interactions with PEPT1. In view of the advantages of nanoparticles in anticancer drug, peptide and protein delivery, it is necessary to explore new speci?c affinity site with intestinal surface. At present, we have successfully prepared L-valine modified doxorubicin-loaded nanoparticles which called transnanoparticles. The oral absorption of doxorubicin was increased by 3.67-fold compared with DOX-Sol in situ intestinal circulation. This project is designed to complete by following experiments: 1. synthesizing PEG-GCDS containing different amino acids（AA-PEG-GCDS）; 2. preparing and evaluating doxorubicin transnanoparticles containing different density of amino acid; 3. using experiments of intestinal absorption, competitive inhibition of hPEPT1, uptake in Caco-2 cells, the inter-cell transportation to study transmembrane absorption mechanism; 4. investigating the pharmacokinitics behaviors and in vivo anti-tumor activity of transnanoparticles. This research is trying to clarify the transport mechanisms of oral nanoparticles targeting PEPT1 and provide a theoretical basis for the transporter as a target for drugs delivery.

当前，纳米粒的非口服给药已得到广泛研究，但口服给药仍是首选的给药方式。寡肽转运蛋白（PEPT1）是肠道转运体的一种，对L-型氨基酸残基具有高度亲和性。将PEPT1作为特异性黏附方式提高纳米粒的口服吸收还未见报道，跨膜转运机制尚不明确。鉴于纳米粒在抗肿瘤药物、多肽、蛋白等口服传递中优势明显，寻找新的肠道黏附方式并明确转运机制是十分必要的。目前我们已成功制备了L-缬氨酸修饰的阿霉素载药纳米粒，大鼠在体肠吸收实验表明其能够将阿霉素溶液的口服吸收提高3.67倍。本课题拟完成：1合成含有不同氨基酸的AA-PEG-GCDS；2制备不同氨基酸密度的阿霉素转运体纳米粒并进行评价；3 通过小肠吸收、hPEPT1 竞争抑制、Caco-2细胞摄取、跨细胞转运实验解析跨膜机制；4考察体内药动学和抗肿瘤活性。通过研究，阐明PEPT1特异性黏附纳米粒提高药物口服吸收的跨膜转运机制，为转运体-纳米粒的构建提供理论依据。

本课题紧紧围绕基于肠道PEPT1特异性黏附的阿霉素口服转运体纳米粒的构建及转运机制开展工作。合成了苄氧羰基(CBZ)保护的CBZ-缬氨酸，聚乙二醇-二硬脂酸甘油酯，CBZ-L-缬氨酸-聚乙二醇-二硬脂酸甘油酯，缬氨酸化化聚乙二醇-二硬脂酸甘油酯的合成，甘氨酰-肌氨酸化聚乙二醇-二硬脂酸甘油酯和甘氨酰－异亮氨酸化聚乙二醇-二硬脂酸甘油酯，构建了基于肠道PEPT1特异性黏附的口服转运体纳米粒所需的载体材料；利用合成的载体材料，制备了阿霉素DS-PLGA自组装的空白纳米粒和转运体纳米粒，并完成了粒径测定，形态观察，包封率和释放度等体外表征；建立了大鼠小肠单向灌流模型，进行阿霉素和空白纳米粒和转运体纳米粒的小肠吸收实验；使用Caco-2细胞模型，通过hPEPT1竞争性抑制实验、细胞摄取实验和细胞转运实验，揭示了进入细胞均属于能量依赖型的，其中空白纳米粒进入细胞是由部分网格蛋白还和部分巨胞饮途径介导的，转运体纳米粒是由部分网格蛋白、部分巨胞饮和部分非网格蛋白和非细胞膜穴样内陷介导途径的；建立了阿霉素大鼠血浆中的血药浓度测定的高效液相色谱色谱质谱连用法并进行方法学验证，应用于阿霉素纳米粒大鼠静脉注射和口服给药的药物动力学研究；建立了C57肺癌荷瘤小鼠模型，进行了阿霉素空白纳米粒和转运体纳米粒的药效学研究。本课题的研究成果为转运体-纳米粒的构建提供了理论依据，为肠道转运体作为药物、多肽、蛋白和基因等纳米载体的口服吸收提供了实验基础。截至到目前，本课题已发表国家级核心期刊1篇，省级核心期刊1篇，现有专利1项和2篇SCI文章正在撰写。