稀土掺杂纳米磷灰石肿瘤显影剂的荧光协同增强与体内代谢

Rare earth doped nano apatite (nREAP) possesses good biocompatibility, biodegradability in intracellular acidic environment and stable fluorescence. It can be used as an imaging agent to achieve the fluorescent bioimaging of tumor in vitro and in vivo, showing a promising potential in diagnosis of tumor. The high fluorescent imaging ability and in vivo metabolism mechanism are the two key problems that are required to be solved for nREAP as the imaging agent of tumor. First, in order to avoid the particle agglomeration and size increasing due to calcination at high temperature as well as the potential toxicity because of increasing doping content of rare earth, a preparation technology of fluorescence synergistic enhancement will be developed to achieve the high fluorescent imaging ability in vivo of nREAP on the basis of energy transfer between rare earth ions, charge compensation and substitution of OH group. Then, in order to avoid the potential risk of radio-labeling method and solve the lower matching performance with metabolism of organic fluorescent labeling method, a quantitative detection method for tracing nREAP in tissues will be developed to clarify the metabolism mechanism in vivo of nREAP on the basis of the Eu(3+) fluorescence quantitative method. Moreover, the surface modification, size and crystallinity controls of nREAP will be studied to achieve the regulation of biodistribution and metabolism in vivo of nREAP. By this study, a biocompatible and biodegradable nano apatite imaging agent with high fluorescent imaging ability can be obtained for diagnosis of tumor. Also, the new idea and method can be provided for investigating the in vivo metabolism of nREAP.

稀土掺杂纳米磷灰石（nREAP）具有良好的生物相容性、细胞内酸性环境可降解性和稳定的荧光性，可作为一种显影剂实现肿瘤的体内外荧光成像，在肿瘤诊断领域展现出良好的应用潜力。高荧光显影能力和体内代谢机制是其作为肿瘤显影剂应用亟需解决的两个关键问题。首先，针对高温煅烧增强荧光造成颗粒团聚长大、提高稀土掺杂浓度引起毒性增加的问题，基于稀土离子间能量传递、电荷补偿、OH基团取代的荧光增强机制，建立一种nREAP的荧光协同增强制备技术，实现其体内高荧光显影能力。其次，针对放射性标记法存在安全隐患、有机荧光物标记法代谢匹配性低的问题，基于Eu(3+)荧光定量分析法，建立一种组织内nREAP的定量检测技术，阐明其体内代谢机制，并通过表面改性、尺寸和结晶度控制来实现其体内分布代谢的可调控。本研究可为肿瘤诊断提供一种生物相容、生物降解的高性能磷灰石类纳米显影剂，为其体内代谢研究提供新思路、新方法。

项目针对稀土掺杂磷灰石肿瘤显影剂应用所需解决的基本问题：荧光成像能力和体内分布代谢，开展了基于稀土离子间能量传递、钠电荷补偿、羟基氟取代荧光增强研究，并进行了表面改性对其水中分散悬浮性的影响研究；通过溶血、细胞毒性以及组织毒性进行了体内外安全性评价；初步评价了荧光成像能力并基于建立的体内定量检测方法开展了磷灰石纳米颗粒体内组织分布代谢的初步研究。基于Eu3+/Gd3+间能量传递荧光效果最大可提高约150%，基于Na+电荷补偿荧光效果最大可提高约90%，基于羟基氟取代荧光效果最大可提升约120%。但是，Na+电荷补偿需要高温煅烧促进Na+的掺入，导致颗粒尺寸长大，这不利于荧光成像的应用，稀土元素共掺和羟基氟取代可采用共沉淀法和水热法进行，尺寸可以保持在纳米级，因此，稀土元素共掺和羟基氟取代两种方式相结合是合适的荧光协同增强途径，荧光效果可提高约270%。进一步减小磷灰石晶粒尺寸到10nm以下，基于晶体场效应和表面效应双重调控，荧光强度相对于沉淀法产物增强约300%，量子产率提高340%，荧光寿命延长450%，有望与稀土元素共掺和羟基氟取代结合进一步提高荧光性能。基于Eu3+荧光定量分析方法，建立了一种稀土掺杂磷灰石纳米颗粒组织含量定量检测方法，为磷灰石基纳米显影剂的体内分布代谢研究提供了技术支撑。基于该方法定量研究了磷灰石纳米颗粒的血液清除和体内组织分布，8h后血液清除率约74%。体内组织分布表现出明显的尺寸依赖性，小尺寸纳米点在肝、脾和肺中的积聚显著减少，分别减少80.2%，79.4%和70.2%，肾脏中纳米点的累积量为大尺寸纳米颗粒的15.4倍，说明网状内皮系统对纳米点清除率降低，纳米点易于通过肾清除。肿瘤中纳米点的累积量为纳米颗粒的5.1倍，被动靶向能力显著增强。因此，尺寸是影响其体内分布代谢的主要因素。