基于自识别系统的中药难溶性成分纳米晶体内行为研究-以槲皮素为例

Nanocrystals (NCs) are internationally recognized as effective methods to address the poor bioavailability of poorly soluble components and greatly enhance the bioavailability of quercetin (QT). However, the key scientific issues about “whether QT-NCs is absorbed as molecules or integral nanoparticles?” and “How much is the contribution both of the molecules and integral nanoparticles for improving bioavailability” still remain unclear..Based on the previous work, this project is aimed at the in vivo biological behavior of QT-NCs and employing the water-quenching fluorescent dyes hybrid technology to prepare fluorescent hybrid QT NCs (QT-HNCs) with different particle sizes. Based on the quenching effect and fluorescence conversion ability, we characterize the structural alterations of the particles to achieve its "visualization" in vivo. A series of Caco-2 cell models (Caco-2, Caco-2/HT29-MTX and Caco-2/HT29-MTX/Raji) and in situ perfusion model are established to investigate the cell uptake and transport and intestinal uptake of QT-HNCs. Lymphatic transportation of QT-HNCs is evaluated by sampling via lymphatic cannulation. The pharmacokinetics and tissue distribution of QT-HNCs after oral administration and injection in rats are also evaluated by IVIS live imaging system and HPLC methods to elucidating the role of particles in enhancing oral bioavailability. This project firstly addresses the fact that the mechanisms of NCs increasing the bioavailability of insoluble components is unclear, resolves the issues of commonness in the industry and promotes the development of traditional ethnomedicine industry.

纳米晶(NCs)是国际上公认的解决难溶性成分生物利用度低下的有效方法，能够大大提高槲皮素(QT)的生物利用度。但是QT-NCs口服后是以分子形式还是以整体纳米粒形式吸收、两者贡献如何的体内行为等关键科学问题仍不清楚。.在前期工作基础上，本项目瞄准QT-NCs体内生物学行为，应用水淬灭荧光探针进行杂化，制备不同粒径槲皮素荧光杂化纳米晶(QT-HNCs)；基于淬灭效应和荧光转化能力，表征结构变化，以实现其在生物体内的可视化；建立细胞模型和单向肠灌流法，研究QT-HNCs肠道摄取及细胞摄取转运情况，肠系膜淋巴插管试验研究QT-HNCs口服经淋巴途径吸收转运；利用活体成像系统和HPLC分别考察QT-HNCs经大鼠口服和注射后体内降解动力学、主要脏器组织分布，阐明粒子在增强口服生物利用度的潜在作用及贡献。本项目首次正视NCs体内行为不清事实，解决行业共性问题，推动民族医药产业发展。

纳米晶(Nanocrystals, NCs)是国际上公认的解决难溶性成分生物利用度低下的有效方法，能够大大提高槲皮素(Quercetin, QT)的生物利用度。但是QT-NCs口服后是以分子形式还是以整体纳米粒形式吸收、两者贡献如何的体内行为等关键科学问题仍不清楚。.本项目瞄准QT-NCs体内生物学行为，应用一种具有遇水聚集淬灭（Aggregation caused quench, ACQ）特性的环境响应型智能荧光探针（简称ACQ荧光探针）进行杂化，采用反溶剂结晶沉淀法成功制备了280nm、550nm、1100nm三种粒径的槲皮素杂化纳米晶（QT hybrid nanocrystals，QT-HNCs）。扫描电镜结果显示，三种粒径的QT-HNCs均呈短棒状；DSC和PXRD结果显示QT原料药及不同粒径的QT-HNCs均为结晶态，表明制备过程及微量ACQ荧光探针的掺入不会影响QT的晶型；体外验证实验显示，QT-HNCs具有较强的荧光稳定性及水淬灭灵敏性，QT-HNCs的药物溶出与ACQ荧光探针的荧光淬灭具有良好的相关性，表明ACQ荧光探针的荧光信号变化能够反映QT-HNCs粒子的整体结构变化，示踪QT-HNCs粒子的体内行为。动物及细胞实验显示，不同粒径的QT-HNCs口服后均有部分以整体QT-HNCs粒子的形式经小肠上皮细胞途径和M细胞转运途径吸收。QT-HNCs粒子与QT药物的吸收和分布存在差异且受粒径的影响显著，小粒径的QT-HNCs-280口服后QT药物吸收快而多，但由于溶出快，QT-HNCs粒子体内保留时间短，保留量少；而中等粒径的QT-HNCs-550口服后QT药物吸收少，但QT-HNCs粒子体内保留时间长，保留量多。两种粒径的QT-HNCs粒子在口服吸收中的贡献分别为37.39%（QT-HNCs-280）和48.89%（QT-HNCs-550）。该研究验证了利用ACQ荧光探针进行NCs体内命运研究的可行性，估算了整体QT-HNCs粒子在口服吸收中的贡献，为NCs粒子的整体吸收提供了有力证据，一定程度上解决了NCs能否整体吸收的争议，对于推动NCs在难溶性中药研究中的发展及临床应用具有重要的意义。