穿膜肽介导的个性化药物直接递送体系设计和穿膜机理研究

Currently, anti-tumor drug is usually delivered by endocytosis process or chemical and physical approaches. However, the endocytosis process leads to the difficulty in drug release from endosome, degradation and excretion, the chemical-physical approaches can result in low delivery efficiency of drug and also cause damage for healthy cells. Hence, it is necessary to develop a novel drug delivery method to improve the drug utilization without cell damage. In this project, a novel cell penetrating peptide (CPP) which is able to efficiently deliver the target drug into tumor cells by direct penetration pathway even at high concentration, will be developed based on the structure of drug using molecular simulations. The penetrating efficiency, penetrating pathway, cytotoxicity and drug delivery of cpp will be determined, and the evaluation system will be established to assess the designed CPPs. The effects of temperature, peptide concentrtion, endocytosis inhibitor and cell membrane receptor on peptide-drug-membrane interactions will be investigated. The mechanism of cellular uptake will be disclosed based on the information of drug delivery efficiency, drug delivery pathway and intracellular drug distribution. Finally, the drug delivery process will be optimized based on the apoptosis of tumor cells, intracellular drug concentration and biosafety to improve the drug utilization. The implementation of this project will provide a guidance for designing the personalized drug delivery system and achieve the drug delievry with high efficiency.

目前，肿瘤治疗药物的递送多采用内吞法和理化机械递送法，其中内吞法存在药物释放困难、降解和外排等问题，而理化机械递送法存在效率低与细胞损伤等问题。鉴于此，本项目针对目标药物的结构特点，采用分子模拟手段设计可介导特定药物直接递呈的穿膜肽。通过细胞实验考察载药穿膜肽的穿膜效率、穿膜方式、细胞毒性和载药性能，构建载药穿膜肽设计评价体系。继而研究温度、肽浓度、内吞抑制剂和细胞膜受体对药-肽-细胞膜相互作用的影响；进一步分析载药穿膜肽的药物递送效率、药物递送途径和药物在细胞内的分布，解析其穿膜机理。最后，以肿瘤细胞的凋亡，胞内药物浓度和生物安全性为评价标准，优化穿膜肽设计和药物递送过程，提高肿瘤细胞内的有效药物浓度。本项目的实施将为穿膜肽介导的个性化药物直接递送体系设计奠定理论基础,实现肿瘤细胞治疗药物的直接高效递送。

目前，肿瘤治疗药物的递送多采用内吞法和理化机械递送法，内吞法存在药物释放困难、降解和外排等问题，而理化机械递送法则有效率低并损伤细胞等问题。鉴于此，本项目通过分子模拟手段设计了可介导紫杉醇高效递呈的穿膜肽R7和Rq。在构建载药穿膜肽设计评价体系基础上，我们考察了不同条件对药物递送过程的影响，验证了其载药穿膜性能、抗肿瘤效果和安全性，解析了其穿膜机理。继而优化药物递送过程，实现目标药物的直接高效递送。细胞水平实验结果表明：1 mM穿膜肽与癌细胞/正常细胞共孵育24 h 未表现出明显细胞毒性，而穿膜肽/紫杉醇和癌细胞共孵育30 min就可以显著抑制其活性，其中在Rq介导下癌细胞内的紫杉醇浓度提高了7倍以上。动物实验表明，在尾静脉注射Rq/PTX后，裸鼠肿瘤生长被有效抑制。进一步的激光共聚焦观察和穿膜机理解析均表明，Rq/PTX的穿膜方式主要为直接递送。本项目建立了面向客户的穿膜肽介导的个性化药物直接递送、实现了肿瘤治疗药物的高效直接安全递送并为药物直接递送过程设计奠定初步理论基础；在肿瘤治疗方面具有很好的应用前景，具有明显的社会经济效益。本项目共发表了3篇论文，其中第一作者SCI 1区和2区期刊论文各1篇；申请了3项专利，培养了3名研究生，圆满完成了预期的研究目标。