Davidiin靶向PECAM-1纳米聚合物口服传递系统及促肠上皮细胞转运机制研究

The result of pharmacology study has shown that Davidiin submits a good anti-tumor activity. But there is obviously known that Davidiin has ellagitannin characteristics of degradation and low oral bioavailability by oral delivery. The key problems are to improve mucosal attachment and enhance GI permeability. Binding of nanocarriers to specific cell surface marker can induce transport into and/or across cell. Based on this, The pAb-(Davidiin-Zein NCs) will be prepared utilized PECAM-1 as a target on GI epithelial cells. This provides internalization of prototype polymer NCs within these cells and transcellular transport by enhancing targeting, uptake,and transport in hunman GI epithelial cell multi-targeted to epitopes of the same receptor. Binding of anti-PECAM-coated polymer NCs to PECAM-1 trggers cell adhesion molecule (CAM)-mediated endocytosis, a pathway distinct from classical transport without apparent alteration of the permeability barrier or cell viability. Fluorescence and electron microscopy, and radioisotope tracing will reveal that anti-PECAM NCs specifically bind to cells in culture, are internalized via CAM-mediated endocytosis, trafficked by transcytosis across cell monolayers without disrupting the permeability barrier or cell viability, and enabled transepithelial transport of polyphenolics. The data come from oral bioavailability and tissue distribution after oral administration of Davidiin in rats will provide more reliable basis. So a new potential way will be shown to improve oral drug bioavailability.

Davidiin具有良好的抗肿瘤应用前景，但口服后体内不稳定，生物利用度低。增加GI粘附和上皮细胞的转运是提高口服生物利用度的关键。纳米粒可通过与GI 表面位点的亲和成分结合，增加口服药物的转运。基于此，我们拟在前期研究的基础上制备靶向PECAM-1的Davidiin玉米醇溶蛋白纳米聚合物，促进药物GI粘附和跨上皮运输，提高化合物的生物利用度。该传递系统利用多克隆抗体的多表位特征，激发细胞粘附因子介导的特殊囊泡内化途径传递药物，不干扰渗透屏障和细胞流动性，明显区别于经典细胞转运路径。我们拟主要采用荧光标记、电子显微镜以及放射性同位素追踪考察pAb-NCs与细胞结合的专属性、经细胞粘附因子介导的内化和经胞吞作用的转运。明确该传递系统在口服吸收后的Davidiin生物利用度和组织分布，为该传递系统特性提供可靠依据。从而为提高口服药物的生物利用度提供一个有效的新途径。

Davidiin具有良好的抗肿瘤应用前景，但在体内不稳定，体内生物利用度低限制了临床应用。研究新型的口服给药系统具有重要的学术意义和潜在应用价值。药剂学研究表明增加GI粘附和上皮细胞的转运是提高口服生物利用度、增加药物疗效的关键。我们假设纳米粒与可靶向GI 表面位点的亲和成分结合可增加药物的转运，因此制备用PECAM-1多克隆抗体pAb修饰包裹的Davidiin纳米聚合物(pAb-(Davidiin-Zein NCs))，考察载体转运行为，探索以PECAM-1为靶点、提高药物的口服吸收利用度的可行性。基于此，本项目组采用体外微生物培养法对中药头花蓼代谢产物Davidiin以及高含量的鞣花酸和熊果酸进行了分离和富集；由于Zein在酸性环境下易于聚集、增大纳米粒直径，不易于药物的释放和吸收，我们优化分离制备酸碱抵抗性强的抗性淀粉，通过pH、微生物影响、塑化剂种类等因素考察了联用抗性淀粉作为口服靶向辅料在胃肠道中增加药物稳定性的能力；采用反溶剂法分别制备了固体纳米粒SP和中空纳米粒HP，所制备的载药HZN粒径约为80nm、包封率60-82%、载药量25.4%；电镜结果显示HZN呈球形，粒径均匀，中心有空洞，壁厚10nm。纳米粒偶联pAb和mAb，以Caco-2细胞和HUVEC细胞为研究对象，利用免疫荧光法定性观察PECAM-1在所选择细胞中的表达情况。结果显示，细胞表面PECAM-1均存在表达，其中HUVEC细胞室高表达。以非靶向纳米粒为对照，考察不同配体修饰度和不同性质的靶向纳米粒的在细胞中的PECAM-1靶向性效果、内涵体／溶酶体逃逸、细胞毒性、细胞摄取机制。细胞毒性试验和细胞摄取结果表明，表面配体密度增加有利于増强HUVEC细胞对纳米粒的抑制和摄取能力；细胞内吞抑制实验表明，纳米粒进入细胞的方式主要是胞饮作用，与PECAM-1结合无明显相关性，受粒径及表面电荷影响大；通过动物体内药代动力学研究，表明中空纳米粒构成的靶向传递系统经口服吸收后，其生物利用度明显高于固体纳米粒。

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