高转膜率肿瘤EGFR靶向的多功能纳米脂质载体的构建与评价

This project is aimed at constructing a tumor EGFR-targeted PEGylated multifunctional nanostructured lipid carrier system(mNLC) coupled with targeting cell-penetrating peptide(CPP) basing on the mild acidic tumor environment. In vitro and in vivo evaluation of this high efficiency membrane-traversing carrier system as well as its mechanism will be performed. R8 is a classic cell-penetrating peptide that has been proved being able to deliver attached bioactive molecules into the cytosolic compartment of cells. In this study, R8 is combined to different degree of PSD-PEG polymerization in order to screen out the optimization that can form a stable conjugate. The targeting efficiency and stability of this R8-PSD-PEG compound under the mild acidic tumor environment will be illustrated through a pH-sensitivity and stability study.An intracellular delivery nanostructured lipid carrier system-mNLC,modified with a tumor homing ligand PE5 and a pH sensitive copolymer R8-PSD-PEG ,will be constructed. The EGFR-targeted peptide ligand PE5 linked to this carrier system is a previous invention of our research group. A thorough comparison between the mNLC and NLC coupled with only one ligand(R8, R8-PSD-PEG or PE5) will be conducted by providing the study of the preparation's physico-chemical property and its stability. Therefore , in vitro and in vivo evaluation of tumor-targeted efficiency , mechanism and therapeutic effect of this mNLC can be demonstrated. Pharmacodynamics research, including measurement of the tumor-suppress efficiency of mNLC and its toxicity on cells and solid tumor will be accomplished using NCI-H1299 cell line and NCI-H1299-transplanted athymic mice. The novel drug delivery system mNLC in this project is supposed to be active targeting and able to increase therapeutic effect without damaging normal tissues. The study of our research group will also show its great potency in active targeting toward bioactive substances such as proteins and peptides . The content of this research is of novelty and feasibility and no similar literature has been published up to now.

本项目拟设计一种基于肿瘤微酸环境的靶向穿膜肽偶联的PEG化的肿瘤EGFR靶向的多功能纳米脂质载体（mNLC），并对其体内外作用及作用机制进行研究。制备不同聚合度的PEG化的多聚磺胺（PSD-PEG），筛选出能与穿膜肽R8稳定结合的PSD-PEG，研究R8-PSD-PEG的pH敏感性、血浆稳定性以确证其肿瘤微酸环境的靶向性；制备mNLC，其中mNLC偶联的靶向肿瘤EGFR的多肽配体PE5为本实验室发明，可以和EGFR特异性结合；对mNLC的理化性质及稳定性进行研究，并将mNLC与单一配体偶联的纳米脂质载体进行系统比较研究，从细胞水平及动物水平评价mNLC的高转膜效率及对EGFR过表达的肿瘤细胞的靶向优势及作用机制、作用效果。本项目研究的mNLC对肿瘤治疗可产生主动寻靶、提高疗效，具有治疗特异性强、不损伤正常组织的优点，具有良好的可行性，研究的内容处于国际前沿，目前尚未见文献报道。

本课题组在依托单位沈阳药科大学的支持下，通过与齐齐哈尔医学院等单位的紧密合作，顺利完成了研究计划，成功制备了利用肿瘤微环境实现“开-关”效应的抗肿瘤药物传递系统PSAR-NLC：以纳米脂质载体（NLC）作为药物载体，在该载体表面修饰特异性识别肿瘤细胞膜上过度表达的表皮生长因子受体（EGFR）的自制小肽AEYLR（2015年获得国家发明专利证书）、具有高效穿膜能力的经典穿膜肽八聚精氨酸（R8）和二硫键-PEG5000（-S-S-PEG），利用二硫键易于被还原剂断裂的性质，实现“开-关”效应, 故将该给药系统命名为PSAR-NLC。。. 在设计之初，首先考察了不同比例R8修饰的NLC（nR-NLC）的制剂学性质、细胞摄取、毒性和在荷瘤小鼠体内的分布情况，对R8比例这一重要参数进行了筛选,结果表明占脂质总质量4%的R8修饰后的4R-NLC具有较明显的提高细胞摄取的作用；在确定R8比例后，在处方中加入另一小肽AEYLR，AEYLR的加入浓度参考了国家自然基金面上项目“新型表皮生长因子受体抑制剂修饰的肿瘤靶向纳米脂质载体的研究”，将AEYLR连接在PEG2000链末端，一方面发挥部分“隐形”作用，一方面确保在R8的存在下能够利用空间效应率先发挥其靶向作用，该两个小肽同时修饰的NLC命名为PAR-NLC，对其细胞摄取、毒性和在荷瘤小鼠的体内分布等特性进行了考察，确保两种肽各自发挥作用的同时产生协同作用，产生类似“1+1>2”的效果。在PAR-NLC上进一步修饰-S-S-PEG2000，制备PSAR-NLC， 在考察其制剂学性质后，在细胞水平和动物水平上测定了PSAR-NLC在还原剂半胱氨酸的存在下具有“开-关”效应，是一种有潜力的抗肿瘤药物传递系统。