基于胸苷酸合成酶靶标和半乳糖凝集素受体介导的多重结肠肿瘤靶向给药系统的构建及评价

Considering the issues existing in the clinical chemotherapy for colon cancer, we intend to develop a class of oral multi-targeted drug delivery system in our project, which is mediated by galectin receptor and then targets at thymidylate synthase (TS).This idea is proposed according to the physiological characteristics of colon and the function mechanism of drugs. The composite film made up by galactosylated chitosan(GC) and sodium alginate(Alg) is degraded in the colon due to a mass of polysaccharide degrading enzymes in this place , thus realizing the positioning release of drug; Simultaneously, this system also take advantage of the properties,including prolonging the duration of this drug delivery system in colon and enhancing the drug uptake by the mucosa cells, which play a role by characteristics of mucoadhesion and promoting the loading-drug through epithelial cells possessed by galactosylated chitosan(GC); Afterwards, the endocytosis mediated by galectin-3 will be utilized to achieve cell targeting for colon cancer, furthermore, the drug enters into the cytoplasm with high efficiency; Ultimately, the metabolites of 5-fluorouracil(5-Fu) and leucovorin(LV) that acts as a synergist for 5-FU will form a triple complex together with TS, contributing to the inhibition of DNA synthesis. The drug delivery system constructed by our project is a three-level target involving colon targeting, colon cancer cell targeting, and TS targeting, and is provided with the characteristics of adhesion to mucosa, consequently shedding light on the design, discovery and druggability of a novel colon cancer targeting therapy with high efficiency and low toxicity.

针对临床结肠癌化疗中存在的问题，根据结肠的生理特点和药物的作用机理,本项目拟构建一类基于胸苷酸合成酶靶标和半乳糖凝集素受体介导的多重结肠肿瘤靶向给药系统。该给药系统利用结肠存在大量多糖降解酶的生理特点，以半乳糖化壳聚糖（GC）和海藻酸钠复合膜降解实现结肠定位释放；同时利用GC具有粘膜粘附性和可能增强所载药物通过粘膜上皮细胞的性质，延长给药系统作用时间，增强药物粘膜吸收；然后利用半乳糖凝集素受体(galectin-3)介导的内吞作用实现结肠肿瘤细胞靶向，并高效入胞到达胞浆；最后利用5-Fu及其增效剂甲酰四氢叶酸（LV）的代谢产物与胸苷酸合成酶形成三联复合物，抑制DNA合成。本项目拟构建的给药系统将集结肠靶向、结肠癌细胞靶向和胸苷酸合成酶靶向三级靶向，同时兼具粘膜粘附性，可为新型高效、低毒的结肠靶向肿瘤治疗的设计、发现和成药性提供理论和实验基础。

本课题组在依托单位沈阳药科大学的支持下，通过与郑州大学等单位的紧密合作，顺利完成了研究计划，成功制备了基于胸苷酸合成酶靶标和半乳糖凝集素受体介导的多重结肠肿瘤靶向给药系统。该给药系统利用半乳糖化壳聚糖载体（GC）对半乳糖凝集素受体介导的内吞作用实现结肠肿瘤细胞靶向，利用5-Fu及其增效剂甲酰四氢叶酸（LV）的代谢产物与胸苷酸合成酶形成三联复合物，抑制DNA合成。. 本课题首先将壳聚糖C6-OH进行半乳糖化修饰，留下了可用于质子化的原壳聚糖结构的 C2-NH2，使构建的纳米载体具有较高的正电荷，从而有利于纳米载体的稳定及细胞的摄取，同时使合成的GC容易通过氨基被进一步修饰，为后续扩展其应用奠定了基础。本课题以半乳糖化壳聚糖为结肠靶向载体构建了三种结肠靶向给药系统，均能较好的主动靶向结肠肿瘤细胞，提高肿瘤细胞的摄取。通过提前添加半乳糖竞争结合结肠肿瘤细胞上的半乳糖凝集素受体，从而降低结肠肿瘤细胞对GC纳米粒的摄取证实了GC结肠肿瘤细胞靶向的机理。. 本课题构建了半乳糖化壳聚糖离子交联纳米粒，并研究表明GC具有和CS相同的黏附性，可以打开细胞间紧密连接，降低跨膜电阻值。课题针对有机纳米粒机械强度较低，物理化学稳定性较差，制备成本较高的缺点，进一步选择介孔氧化硅作为纳米粒的载体材料，以GC修饰，构建的MSN纳米粒和未修饰的载体相比，负载5-FU和亚叶酸钙（LV）均具有较高的载药量，为难溶性载体高效负载水溶性小分子提供了新的途径。. 静脉滴注或推注5-FU/LV已成为全世界结肠癌化疗的一线治疗方案，其与单独给5-FU相比，能够显著改善患者存活率，但LV给药系统的研究较少。本课题首次构建了LV的MSN纳米粒，并证实其能增加5-FU@MSN-NH2/GC对SW620细胞的抑制作用，其机理可能和能够显著下调SW620细胞中的TS表达水平有关，为LV的制剂研究及其对5-FU制剂的增效研究提供了新的思路和途径。