叶酸受体靶向的超顺磁/荧光/VEGF siRNA多功能硅纳米载体的抗肿瘤作用及其成像规律研究

Targeted and controlled drug/gene delivery systems are called "mystic pharmaceutics". Angiogenesis is the formation of new blood vessels from preexisting microvessels. In particular, tumor growth and metastasis were found to be dependent on angiogenesis. Thus, anti-angiogenic therapy has become an important route for cancer treatment. This strategy for anti-cancer therapy has many excellent traits, for instance, good effect, low toxicity, and almost no drug resistance. Many literatures reported that anti-angiogenesis by inhibition of VEGF has been considered as a promising approach to suppress growth and metastasis of malignant tumors. However, the use of siRNA in clinical applications is still questioned because of its inherent instability in biological fluids along with poor or non-specific cellular uptake. To address these problems, non-viral delivery systems based on nanoparticles have been introduced for siRNA. In the present research proposal, a new and multifunctional delivery system for VEGF siRNAs will be developed. The multifunctional carriers are core-shell structure (Fe3O4@SiO2), and fluorescent dye molecules (FITC) were doped into the silica shells. Then surface modification will be further carried out for conjugating folate and VEGF siRNA to form a novel type of multifunctional nanoparticle complexes (referred as to Fe3O4@SiO2[FITC]-Folate/VEGF siRNA). The Fe3O4@SiO2[FITC]-Folate/VEGF siRNA has the capability of specific accumulation in the folate receptor-positive cancer cells. The anti-angiogenic activity and tumor growth inhibition activity of the Fe3O4@SiO2[FITC]-Folate/VEGF siRNA complexes were evaluated after intratumoral or intravenous administration in an animal tumor model. After systemic injection of Fe3O4@SiO2[FITC]-Folate/VEGF siRNA complexes, bio-distribution profiles in various tissues were quantitatively determined and visualized. In addition, The NPs endocytosis mechanism and fluorescent imaging were also investigated. The proposed research will provide more experimental information for its clinic applications in the future.

靶向可控的药物/基因纳米载体被称为"梦的制剂"。抗血管生成疗法以其效果稳定、不易产生耐药性、毒副作用小等特点，成为肿瘤治疗研究的热点之一。本项目以构建叶酸受体靶向的超顺磁/荧光/VEGF siRNA多功能硅纳米载体选择性识别肿瘤细胞表面特定高表达的分子标记物叶酸受体（folate receptor, FR），提高对肿瘤细胞(或组织)的选择性识别和在靶细胞上可控聚集为出发点，深入而系统地研究此纳米载体系统的合成条件、特性表征、输送效果、细胞内分布规律、纳米载体代谢规律（动力学）、目标蛋白VEGF表达抑制效果及肿瘤血管新生抑制情况，以此来评价纳米基因载体的靶向治疗效果。然后利用干扰细胞内吞途径的特异性抑制剂，分析和探讨此多功能硅纳米基因输送载体被肿瘤细胞内吞的分子机制与转运机理，同时建立动物模型，重点分析在体条件下的抗肿瘤效果和荧光成像规律，为其最终的临床应用提供实验数据和参考信息。

药物/基因控释系统的研究已成为纳米医学领域的一个研究热点，而药物或基因载体是目前药物靶向输送和基因治疗能否成功的关键环节之一。由于纳米载体具有一些独特的优点，因而可以在疾病的诊断和治疗中发挥重要作用。制备具有化疗、基因治疗、多药联合应用以及诊疗一体化的多功能性抗肿瘤药物输送系统是未来纳米医学的发展一个重要方向。该项目以叶酸受体(FR)为目标分子，通过对超顺磁/荧光二氧化硅纳米粒子表面进行表面修饰，使之带有叶酸和VEGF siRNA，从而可以特异地识别肿瘤细胞表面FR，达到主动靶向治疗和成像的目的。基于此，深入探讨载体对肿瘤细胞输送效果、跨膜转运、代谢动力学、目标蛋白表达抑制效果和肿瘤血管新生抑制情况，研究了载体内吞与转运的机制，重点分析此纳米载体的抗肿瘤效果和荧光或核磁共振(MRI)成像规律(Expert Opin Drug Del. 2012, 9: 1197-1207; Int J Nanomedicine. 2013, 8: 1897-1906; J Control Release. 2013, 172(1): e120-e121; Sci Rep. 2014， 4: 7072; ACS Appl Mater Interf. 2014, 6: 21615-21623; Int J Nanomedicine. 2015, 10: 4279-4291)。主要研究结果如下：(1) 成功制备了能够高效运输VEGF siRNA并可作为MRI造影剂的复合纳米载体Fe3O4@SiO2/PEI/VEGF siRNA，载体呈均匀球型，核-壳结构明显，具有较高的磁饱和强度、低细胞毒性和良好生物相容性，该载体能够很好地被HeLa细胞内吞并明显地抑制了VEGF表达，MRI成像效果良好，表明该载体是一种高效的诊疗一体化纳米载体，可用于肿瘤的治疗与诊断。(2)构建叶酸(FA)和FITC共修饰的壳聚糖(FA-CS-FITC)复合分子，通过自组装方式实现化疗药物阿霉素(DOX)、碳量子点(C-Dots)和VEGF siRNA的共载，其大小均一，生物相容性好、高稳定性、pH响应性等优点。生物学实验证实，复合纳米药物FA-CS-FITC(DOX/C-Dots)/VEGF shRNA具有很好的协同抑制肿瘤细胞增殖(高达74%)，促进肿瘤细胞凋亡，提高肿瘤治疗效果的能力，同时具有细胞示踪与成像功能，