超声介导的"治疗诊断"多功能超分子基因微载体的研究

Although the principle of cancer gene therapy has been proved for a long time, there are still many challenges for its clinic application. Armed at the key issue of cancer gene therapy, a new strategy of ultrasound-assisted cancer gene therapy will be explored via the combination of the clinic-realizable ultrasound microbubble technology and the non-viral gene vectors in this research. The multifunctional microcarriers will be specially designed by host-guest assemble of the cyclodextrin-modified gene nanoparticles and targeting ligands onto the ferrocene-modified PEGylated microbubbles. We hypothesize that the multifunctional microcarriers will circulate sufficiently long due to the PEG shell and accumulate in the tumor tissue due to the targeting ligands. Real-time ultrasound diagnostic imaging can be obtained to identify the tumor sites. Microbubble-induced sonoporation can be used as the driving force to transfer gene vectors into cells by improving the permeability of different biofilm, which thus lead to efficient transfection. The synergistic effect of ultrasound stimulus and gene transfection will be explored as a new approach to enhance the in-situ gene transfection. The potential of the novel multifunctional microcarriers in ultrasound-assisted cancer gene therapy will be investigated.

本课题针对癌症基因治疗的关键问题，将可临床实现的超声微泡技术与非病毒基因载体相结合，制备表面二茂铁修饰的PEG化微泡造影剂，并通过主客体超分子组装，将环糊精修饰的靶向基团及基因纳米粒子组装到微泡造影剂的表面，构筑非病毒基因载体/微泡超分子组装体。利用PEG的长效循环作用和靶向分子的识别作用，实现功能微载体在癌症组织的高效富集；利用微泡的超声成像功能实现对病变组织诊断成像；利用超声"声孔效应"实现非病毒基因载体的多种生物膜跨越传递；利用非病毒基因载体实现基因的高效转染。深入研究这种多功能集成设计方法对延长超声微泡体内循环时间和增强靶向富集功能的影响，探索超声刺激和基因转染协同增强的基本规律，为建立超声辅助癌症基因治疗手段提供切实可行的途径。

本项目针对癌症基因治疗的关键问题，将可临床实现的超声微泡技术与非病毒基因载体相结合，构筑非病毒基因载体/微泡超分子组装体，实现了超声刺激下基因转染的显著增强。首先以临床商用的声诺维微泡，分别采用三种不同性质的非病毒基因载体：聚乙烯亚胺（PEI）、聚赖氨酸（PLL）、含双硒PEG化聚乙烯亚胺（PEI-SeSe-PEG），采用体外细胞培养，探索超声辐照参数（如超声能量、辐照时间等）对细胞活性及基因转染的影响。结果表明：由于超声微泡产生的“声孔效应”，有利于基因直接进入细胞质甚至达到细胞核，显著提高基因转染效率；制备阳离子化白蛋白基因载体，研究了阳离子链段长度、接枝率对基因转染效率的影响，结果表明：BSA-PEI1800上的PEI1800具有“质子海绵效应”，有助于溶酶体逃离，因此具有更高的转染效率，与PEI25K相当；构建基于阳离子白蛋白BSA-PEI1800的微泡，进一步采用静电组装制备微泡@基因超分子组装体；分别制备环糊精修饰的聚乙烯亚胺和二茂铁修饰的脂质体微泡，构建了基于主客体组装的非病毒基因载体/微泡超分子组装体。探索利用超声“声孔效应”实现非病毒基因载体的多种生物膜跨越传递，实现了超声刺激下基因转染的显著增强，为建立超声辅助癌症基因治疗手段提供切实可行的途径。基于以上研究已发表SCI论文16篇，另有3篇文章正在投稿中。