利用磁垂钓技术研究蛋白纳米笼在活体内的蛋白冠效应

Protein nanocages are cage-like protein nanostructures self-assembled from one or a few kinds of subunits, for example, ferritins and virus-like particles (VLPs). As natural supermolecular biomaterials, they have shown unique advantages and application potentials in the fields of imaging, vaccine, drug delivery, theranostics and tissue repair. However, unclarity of mechanism of their in vivo biological effects, difficulty in controlling their in vivo behaviors and poor targeting have greatly impeded the development of protein nanocage-based biomedical applications. Aiming to address the fundamental question, that is, how the fate of protein nanocages is determined once they enter the living body, this proposal plans to establish a magnetic fishing assay with the VLP of simian virus 40 (SV40) as a model, by encapsulation of a superparamagnetic iron oxide (SPIO) nanoparticle inside the cavity of VLP. With the aid of such an assay, the proteome (as the fish) that binds to the surface of SV40 VLP (as the bait) in vivo, termed protein corona, will be separated conveniently and analyzed qualitatively and quantitatively. The time- and organ-dependent changes of composition of protein corona will be revealed. And the role of protein corona in determining the in vivo fate of SV40 VLP will be assessed. This project would offer new insights into the molecular mechanism of in vivo biological effects of protein nanocages, as well as a novel research strategy. The findings of this project might also serve as basis and guidance for both regulation of in vivo behaviors of protein nanocages and future design of targeted nanodevices. The efforts of this project are expected to help to overcome the obstacles to biomedical applications of protein nanocages.

蛋白纳米笼是由一种或几种蛋白亚基自组装形成的笼形结构（如铁蛋白、病毒样颗粒等），是天然超分子生物材料，在成像、疫苗、药物递送、一体化纳米诊疗、组织修复等领域展现出独特优势和应用潜力，但是其活体生物学效应机制不明确、活体行为难以调控、靶向效率低等问题严重阻碍了相关生物医学应用的发展。本项目围绕“蛋白纳米笼进入活体后的命运决定”这一基本问题，以SV40病毒样颗粒为例，通过自组装在其内腔包装超顺磁性纳米粒子，建立活体磁垂钓技术，进而对病毒样颗粒（“饵”）在活体中的表面结合蛋白质组（“鱼”），即蛋白冠（protein corona），进行磁分离和定性定量研究，揭示蛋白冠组分种类与丰度的时空变化规律，解析蛋白冠在病毒样颗粒活体命运决定中的作用，为深入认识蛋白纳米笼生物学效应的分子机制提供新的视角和研究策略，为蛋白纳米笼的活体行为调控与靶向器件设计提供依据和指导，为扫除其生物医学应用障碍打下一定基础。

蛋白纳米笼（protein nanocage, PNC）因其可控组装、结构可寻址、易于再设计、生物相容性好等特点，作为天然生物纳米材料在生物成像、疫苗开发、靶向递送、组织修复等领域表现出了独特的应用价值。血液蛋白冠为研究PNC活体效应与行为规律、突破活体应用瓶颈提供了重要视角。本项目严格按照任务书既定目标、内容和方案，开展了相关研究，取得如下进展：建立了基于PNC临界浓度控制原理的外源物质无损包装策略；通过可控分子自组装，成功在PNC的内腔包装超顺磁性纳米粒子，建立了PNC血液蛋白冠的磁垂钓技术；以PNC的肿瘤靶向为研究体系，采用不同表面修饰方案，获得了活体水平高效肿瘤靶向的PNC，并系统分析了活体中PNC表面的血液蛋白冠组成，发现结合更少的补体相关蛋白和更多的载脂蛋白有利于延长PNC的血液循环时间和提高肿瘤靶向效率，揭示了蛋白冠在PNC活体命运决定中的重要作用。该项目所建立的纳米磁垂钓技术，将为蛋白类纳米材料的活体行为研究提供新的技术模式，相比传统方法，提高了蛋白冠的分离纯化的效率、纯度和保真度，同时也为病毒学中受体发现、病毒-宿主相互作用研究提供了新方法。相关成果对PNC的活体行为调控与靶向生物纳米器件设计也具有重要的指导意义。