双重介导脂质体脑靶向给药系统的研究

The exist of blood-brain barrier(BBB) restrained lots of drugs to be applied in the treatment of central nervous system(CNS) diseases. A drug delivery system targeting to the brain would provide a promising prospect to the drugs which have been used in clinic. On account of the construction features of BBB, we designed a novel dual-mediated liposomal delivery system targeting to the brain, involving in absorptive-mediated and receptor-mediated. . In the project, we would design and synthesis a series of cell penetrating peptids(CPP) according to a Phage Display-Based High-Throughput Screening System and screen them on the model of BBB in vitro to obtain a high-penetrating CPP which was called PTB(peptide targeting to brain).OX-26, a monoclonal antibody to transferrin receptor on BBB, acted as receptor-mediated ligand here. Furthermore，we would investigate the density of each peptide used in PTB/OX26-PEGylated liposomes by using Flow cytometry(FCM), and then make sure of the formulation of the dual-mediated liposomes. The qualities of PTB/OX-26-PEGylated liposmes would be estimated before its transport mechanism was studied on the BBB model. In vivo studies, pharmacokinetics as well as bio-distribution and accelerated blood clearance(ABC) phenomenon were observed by using 99mTc labelled lipsosomes. Pharmacodynamics of doxorubicin encapsulated into the liposomes would be studied on the rat model of C6 glioma. . The project planned to develope a novel drug delivery system with high stability, transport efficiency and selectivity targeting to the brain as a result of systemic study both on cell level and in vivo level. The novel dual-mediated liposomal delivery system would establish a theory foundation for the treatment of the CNS diseases.

血脑屏障（BBB）的存在限制了药物在中枢神经系统（CNS）的应用，脑部给药系统的研究有望为目前已有的CNS药物提供广阔的应用前景。本课题基于BBB的结构特点，设计了受体介导和吸附介导双重修饰的脂质体作为脑靶向新型药物传递系统。以噬菌体展示技术高通量筛选得到的TAT-PTD肽段序列为基础，应用多肽合成技术和BBB体外模型，设计合成筛选得到高BBB穿透性细胞穿透肽PTB；应用流式细胞技术研究双重介导脂质体PTB/OX-26-PEG-L中两种导向分子的密度，确定处方和制备方法，进行质量评价；应用BBB模型通过干预实验探讨其转运能力和转运机制；体内研究99mTc标记脂质体药动学、组织分布以及ABC现象；应用大鼠C6脑胶质瘤模型，评价包载阿霉素脂质体的药效学。本项目力求通过细胞水平和在体水平的系统研究，得到高稳定性、高转运效率、高选择性的脑靶向给药系统，为CNS疾病的治疗提供理论依据和研究基础。

血脑屏障（BBB）的存在限制了药物在中枢神经系统（CNS）的应用，脑部给药系统的研究有望为目前已有的CNS药物提供广阔的应用前景。.本课题基于BBB的结构特点，设计了通过受体和吸附介导两种途径，双重分子修饰的脂质体作为脑靶向新型药物传递系统。以狂犬病毒受体蛋白与九聚精氨酸偶联物（RVG29R9）为模板，在计算水平应用分子动力学模拟等方法设计得到了5种与乙酰胆碱受体具有高亲和力的多肽，并用固相合成技术得到了纯度高于95%的多肽。经BBB体外模型通透率测定，优化得到了RVG4，其通透率系数为3.45*10-4 cm/s，远高于其他RVG系列，是RVG29R9模板的1.3倍，是R9的5.48倍。结果表明RVG4具有较高的BBB通透性，是一种具有潜能的靶向BBB的细胞穿膜肽。.应用流式细胞技术研究双重介导脂质体Tf-CPP-SSL中两种多肽的密度，确定处方和制备方法，进行质量评价。结果表明最优Tf-CPP-SSL中Tf和CPP分别通过长链PEG3400和短链PEG2000修饰于脂质体表面，修饰密度分别为1.8%和4%。其粒径约为100 nm，具有较好的稳定性，BBB通透性，无溶血性。Tf-CPP-SSL跨BBB转运机制为首先通过Tf受体介导拉近了CPP与BMVEC的距离，然后在乙酰胆碱受体介导和网格蛋白的介导下，进入细胞。进入靶细胞后，CPP在溶酶体中质子化，与带负电的溶酶体膜发生静电相互作用，促进膜融合，引起药物从溶酶体中的逃逸，提高了其跨膜转运能力。.体内药动学研究结果表明，与SSL比较，CPP-SSL从血液中消除速率明显增加，体现了CPP对正常组织的穿透能力，但双重修饰脂质体 Tf-CPP-SSL具有长循环特征，降低了药物在非靶器官的聚集。24小时,Tf-CPP-SSL在肿瘤组织的摄取是SSL的8倍，体现了对肿瘤的高选择性。药效结果表明Tf-CPP-SSL（DOX）组显著改善了动物体重的降低，提高了生存率。给药15天后，Tf-CPP-SSL,Tf-SSL和SSL的抑瘤率分别为87.13%，62.17%，31.3%。.本项目将计算化学和药剂学相结合，设计筛选得到了一种对于BBB具有高穿透性的PTB，通过细胞水平和在体水平的系统研究，构建了一种高稳定性、高转运效率、高选择性的脑靶向给药系统，为CNS疾病的治疗提供理论依据和研究基础。.