细胞转运肽和两亲性壳聚糖衍生物修饰纳米粒用于提高蛋白多肽药物口服结肠吸收的研究

Colon is an ideal absorption site of protein and peptide drugs. However, some barriers, such as the barriers of diffusion and mucosal permeability, may obstruct colonic absorption of proteins and peptides. In order to improve colonic absorption of proteins and peptides, the nanoparticles modified with cell transport peptides (CTP) and amphipathic chitosan derivatives (a-CS) are designed as the vehicle for the colon delivery of proteins and peptides in the research project. It possesses several advantages. For example, CTP on the surface of the nanoparticles, with the activity of transepithelial delivery, might mediate the colon delivery of nanoparticles containing proteins or peptides, which might overcome the barrier of mucosal permeability. Additionally, the mucoadhesion of the nanoparticles modified with a-CS, a bioadhesive material, might reduce the barrier of diffusion towards the absorptive mucosa. Furthermore, mucoadhesion to the colonic mucosa could normalize the transit time of drugs and allow more consistent performance of formulations within and between individuals, improving the overall efficacy of drugs. In the plan of the research project, some factors, which may effect on the mucoadhesion of nanoparticles, colonic microbial-triggered degradation of a-CS on the surface of nanoparticles, and the protection of a-CS against the degradation of the CTP in gastrointestinal tract, will be investigated systematically. The transport of modified nanoparticles in Caco-2 and ex vivo model, the pharmacodynamics of nanoparticles containing insulin after oral administration and the transit of nanoparticles in the gastrointestinal tract of beagle dogs will also be carried out. The study of the research project may provide theory basis and a feasible measure for the clinical application of CTP in oral protein and peptide drug delivery. The project is an extensive and deeper study of our researching program. It is novel, innovative, valuable, and highly worthy of accomplishing.

结肠是蛋白多肽类药物口服较理想的吸收部位，但存在扩散和穿透等屏障。为提高蛋白多肽口服结肠吸收，本课题采用细胞转运肽（CTP）和两亲性壳聚糖衍生物（a-CS）共修饰的纳米粒作为蛋白多肽口服给药的载体，通过CTP修饰增加纳米粒跨肠细胞的转运，克服蛋白多肽结肠吸收的穿透屏障，通过a-CS修饰赋予纳米粒较好的生物粘附性，克服载药纳米粒在肠腔中的扩散屏障。此外，结肠生物粘附还能延长吸收时间，并使滞留时间标准化，在提高吸收的同时减少个体差异。本项目将系统研究影响修饰纳米粒的生物粘附性、a-CS酶触发降解性能和在胃肠道中对CTP保护作用的各种因素；修饰纳米粒在肠道细胞和离体模型中的转运及其机制；载药修饰纳米粒动物体内药动学及在消化道不同部位的运行行为，为CTP在提高蛋白多肽口服吸收中的实际应用创造条件并提供理论依据。本课题是申请者在研项目的延续和深化，设计思路新颖，具有较强的创新性和较大的研究价值。

本项目设计制备了细胞转运肽（CTP）和两亲性壳聚糖衍生物（a-CS）共修饰的纳米粒作为蛋白多肽的给药载体，以提高该类药物的口服结肠吸收。首先，合成了四种a-CS，即辛基、十二烷基、软脂酰基或硬脂酰基化的N-三甲基壳聚糖衍生物（TOCS、TDCS、TPCS、TSCS）并对合成产物进行表征。然后，以a-CS为乳化剂，通过处方和工艺优化，制备a-CS单修饰纳米粒（CS-NPs）：TOCS-NPs、TDCS-NPs、TPCS-NPs和TSCS-NPs，以及a-CS和CTP（Tat、Penetratin和R8）共修饰纳米粒（CTP-CS-NPs）：Tat-TDCS-NPs、Pen-TDCS-NPs、R8-TDCS-NPs、Tat-TOCS-NPs、Tat-TPCS-NPs和Tat-TSCS-NPs，并对纳米粒体外性质进行了研究。对a-CS在结肠环境中的降解行为的研究表明，a-CS在结肠酶作用下迅速降解。考察了a-CS对修饰纳米粒表面CTP在胃肠环境中的保护作用，结果表明，在含胰蛋白酶的人工肠液中，Tat-TDCS-NPs对Tat的保护作用优于以泊洛沙姆为乳化剂制备的纳米粒。粘蛋白试验和大鼠离体实验证明a-CS修饰纳米粒呈现良好的生物粘附性。Caco-2细胞模型实验结果表明，相比以PVA为乳化剂制备的纳米粒（PVA-NPs）和TDCS-NPs，CTP-TDCS-NPs的细胞摄取和转运均显著增加，尤其是Tat-TDCS-NPs，在4h内的药物摄取量分别是PVA-NPs的3.68倍和TDCS-NPs的2.3倍，药物转运量分别是PVA-NPs的2.84倍和CS-NPs的1.56倍。糖尿病大鼠模型药效学实验结果表明，装载模型药物胰岛素的Tat-TDCS-NPs降血糖效果分别是PVA-NPs的2倍和TDCS-NPs的1.5倍，持续时间达8h。进一步选用巴马香猪作为动物模型进行药动学和药效学评价，结果表明，与TDCS-NPs相比，Tat-TDCS-NPs的AUC和Cmax分别提高1.45倍和1.82倍，胰岛素Tmax为12h，最大降血糖效应也出现在12h，推测该纳米粒到达结肠后发挥作用。通过对巴马香猪灌胃99mTc同位素标记的Tat-TDCS-NPs，采用单光子发射计算机断层成像术（SPECT）在不同给药时间对其胃肠道扫描示踪成像，进一步证实Tat-TDCS-NPs能够到达结肠发挥作用。