D型IC-8多肽修饰的还原敏感型RHB自组装双靶向核酸递送载体的研究

To increase the targeting, stability, security and efficiency of non-viral gene delivery vector, we design an efficient, stable, low toxicity and dual-targeting ternary complex nanoparticles as non-viral gene delivery system. Our interest has been focused on a redox-sensitive dendritic poly amino-amine (RHB) as carrier material which condense DNA or siRNA self-assembly into nano-complexes. We hypothesized D-IC-8 peptide which target to the Annexins receptor of tumor vascular endothelial cells modified hyaluronic acid (HA) coated RHB/DNA or siRNA complexes to form CD44 targeting and tumor vasculature targeting dual targeting core-shell redox-sensitive IC-8-PEG-HA/RHB/DNA or siRNA ternary complexes system based on electrostatic self-assembly adsorbed coatings. The screening and optimization of the system formulation were performed with modern pharmacy technology; the complexes were characterized by small-angle X-ray diffraction on two-dimensional structure; the ways and mechanisms of the cellular uptake would be clarified; we will use confocal microscopy to examine the intracellular fate and targeting. The transfection efficiency and targeting of the system will be evaluated on the cellular and whole animal level.

针对非病毒基因递送载体的靶向性、稳定性、安全性和高效性等关键问题，设计并构建高效、稳定、低毒的双靶向三元复合物非病毒载体。本课题拟采用还原敏感型超树枝化聚胺基-胺 (RHB) 作为载体材料与质粒DNA或siRNA自组装压缩成纳米级复合物，以靶向肿瘤血管上皮细胞Annexins受体的D型IC-8肽修饰的透明质酸(HA)对该复合物进行静电包衣，形成靶向肿瘤血管和肿瘤细胞CD44的双靶向还原敏感性IC-8-PEG-HA/RHB/DNA或siRNA三元复合物系统。采用现代药剂学技术对该系统的制备方法进行筛选和优化；利用小角度X衍射对三元复合物的二维结构进行表征，阐明该系统进入细胞的途径和机制；采用激光共聚焦技术和活体成像技术考察该系统在细胞内的转运情况和肿瘤靶向性。从细胞水平和整体动物水平评价该载体系统的转染效率和靶向治疗肿瘤的应用效果。

课题通过药剂学、高分子材料学和分子生物学等技术手段的交叉应用，基于静电包衣策略成功构建了RIF7-HA/RHB/DNA双级靶向三元复合物基因药物非病毒递送载体。采用HA静电包衣后，HA/RHB/DNA复合物的细胞毒性降低。不同质量比的复合物在肿瘤细胞中的转染能力显示HA/RHB/DNA复合物的转染效率高于二元复合物；3D肿瘤球实验结果表明三元复合物具有良好的渗透性并能够成功转染。HA能显著提高RHB/DNA在体内肿瘤的转染效率，三元复合物的目的蛋白表达量是二元复合物的2倍。构建的靶向CD44的anti-P123-PPI/DNA非病毒递送载体体内研究结果表明靶向CD44的基因递送载体设计可有效提高其体内肿瘤靶向性和有效性。三元复合物的入胞途径主要通过网格蛋白、穴小泡和巨胞饮三种内吞途径进入细胞，并能有效转染，证明了复合物经内酶体-溶酶体系统向细胞核转运。通过逆序D型改造合成了RIF7多肽，显著提高多肽的体内稳定性，并且能够保持对Anxa1受体的靶向特异性。经过RIF7多肽修饰后，叠加了RIF7对Anxa1受体与HA对CD44受体的特异性结合，细胞摄取量达到(73.25±4.64)%，进一步提高复合物的细胞转染效率。靶向三元复合物RIF7-HA/RHB/siRNA给药后24 h即出现明显的沉默虫荧光素酶表达效果，48 h进一步增强，直观地反映了复合物体系在肿瘤部位的蓄积过程，可以高效的将治疗基因成功递送到肿瘤部位并成功完成体内转染。IF7-HA/RHB/DNA双级靶向三元复合物基因非病毒递送载体对改进现有基因药物递送系统具有非常重要的指导意义；也为实现核酸药物有效体内递送提供思路和理论基础。