病毒样纳米颗粒的长期生物安全性评价研究

Virus based nanoparticles (VNP) or virus like nanoparticles (VLP) typically consist of several hundreds to thousands of protein molecules, which self-assemble to form a hollow scaffold packaging without the viral nucleic acid. For their great advantages of morphological uniformity, biocompatibility and easy functionalization, virus based nanoparticles originated from a variety of sources are being developed for biomedical and nanotechnology applications that include tissue targeting and drug delivery. However, several issues remain illuminate where the use of viruses as materials (for biomedicine in particular) has a few hurdles to overcome, such as the further studies of the toxicity, immunogenicity and bio-distribution of virus particles in vivo, which will provide important information regarding the safety and potential efficacy of the particles in a therapeutic situation. Herein, in this proposal, we will choose cowpea mosaic virus (CPMV), tobacco mosaic virus (TMV) and simian virus 40 (SV-40) as objects of study. Comparing to the different of protein molecules, different doses, different size and different surface modifications of viral based nanoparticles, we will study the effect of these nanoparticles on biological molecules in blood and tissues. And we will also further study the toxicity, immunogenicity, immunoresponding, bio-distribution, storage, transporting and metabolism of virus based particles in vitro and in vivo after a long time exposure. Based on these studies, we attempt to summarize whether the toxicity effects in vivo originated from the long time exposure of viral based nanoparticles or not. And we attempt to summarize the relationship between toxicity or immunoresponding of viral nanoparticles in vivo and some characters of viral componets, particle size or surface modification after a long time exposure. The planning study will illuminate the mechanism for bio-effects and toxicology of viral based nanoparticles in vivo or in vitro. Also, it will lay the foundation of developing viral based nanoparticles for biomedical and nanotechnology applications.

病毒样纳米颗粒是一类无病毒基因组、由衣壳蛋白自组装而成的重要生物纳米材料，其颗粒均一、可修饰、生物相容性较好，被广泛用于药物递送载体、医学成像诊断和治疗等研究。然而，其体内长期毒理学效应和生物安全性评价仍处于空白，严重束缚了它们在临床医学上的应用和开发。鉴于此，本项目拟以豇豆花叶病毒、烟草花叶病毒和猴空泡病毒-40三种代表性病毒样纳米颗粒为研究对象，通过考察其不同蛋白组分、剂量、尺寸大小和比表面性质差异，全面解析它们与相关生物效应分子的相互作用、细胞毒性和小鼠体内分布、蓄积、代谢及免疫应答关系等长期毒理学生物效应。深入阐明和揭示病毒样纳米颗粒体内长期暴露是否会引起毒理学效应、其体内免疫应答与病毒颗粒物组装组分和包装组分之间的关系、以及它们的纳米特性与体内长期生物安全性的关系。本项目的开展将充分阐释病毒样纳米颗粒的生物效应和纳米毒理学机制，也将为病毒纳米材料的医学应用发展提供强有力的保障。

病毒样纳米颗粒是一类仅由衣壳蛋白自组装而成的重要生物纳米材料，其颗粒均一、可修饰、生物相容性较好，被广泛用于药物递送载体、医学成像诊断和治疗等研究，然而，其体内长期毒理学效应和生物安全性评价仍处于空白。本项目利用细胞和多种小鼠动物模型，以SV40、TBEV以及EBOV等不同组分、不同修饰分子和不同包装物组成的病毒样颗粒为研究对象，研究它们在体内的长期暴露是否会引起毒理学效应，探讨这些颗粒物的纳米属性和组分是否引起毒理学效应，阐明这些病毒样颗粒物在体内的长期生物安全性。主要结果：第一，构建和建立多种野生型和荧光标记的SV40、TBEV和EBOV等病毒样颗粒，形态学和荧光性质表征结果表明，病毒样颗粒的形状、尺寸与对应真病毒吻合，荧光性质稳定。第二，细胞水平实验表明SV40病毒样颗粒和材料对细胞无毒性，具有较好的生物安全性，TBEV和EBOV病毒样颗粒对细胞有一定的毒性和损伤。第三，利用SV40病毒样颗粒靶向小鼠动脉粥样斑块、乳腺瘤、结肠癌和脑胶质瘤等为研究模型，监测长期生物安全性，结果表明SV40病毒样颗粒这一类具有较好载带功能的病毒材料均无明显的毒理学效应，长期生物安全性较好。第四，利用TBEV病毒样颗粒与小鼠血脑屏障的相互作用，以及EBOV病毒样颗粒与小鼠血-视网膜屏障相互作用为研究模型，探讨了这两类病毒样颗粒的长期生物安全性，结果表明TBEV对血脑屏障有一定的损伤，提示是由于TBEV主要组成组分的E蛋白对血管内皮细胞造成损伤；EBOV病毒样颗粒也对小鼠血-视网膜造成损伤，提示组成蛋白之一的GP蛋白对该体系造成损伤，深入研究表明GP蛋白通过刺激周细胞分泌内皮细胞生长因子破坏血-视网膜屏障。综上，生物型病毒样颗粒纳米材料的纳米属性不会影响其生物安全性，SV40等病毒样颗粒有较好的长期生物安全性，TBEV和EBOV等存在多组分蛋白的病毒样颗粒具有一定的毒理学效应，长期生物安全性较差。